Genetic modification of endothelial cells

ABSTRACT

Endothelial cells transduced with genetic material encoding a polypeptide or protein of interest and, optionally, a selectable marker, as well as methods for making and using the transduced endothelial cells are disclosed. Such endothelial cells are useful in improving the performance of vascular grafts and in delivering the encoded polypeptide or protein, such as an enzyme, a hormone, a receptor or a drug, to an individual.

SPONSORSHIP

Work described herein was supported by grants from the NationalInstitutes of Health, the Howard Hughes Medical Institute, and theWhitehead Institute for Biomedical Research.

RELATED APPLICATION

This is a continuation of application Ser. No. 07/786,188, filed Oct.31, 1991, now abandoned, which is in turn a continuation-in-part ofapplication Ser. No. 07/607,252, filed Oct. 31, 1990, now abandoned,which is in turn a continuation-in-part of Ser. No. 07/131,926, filedDec. 11, 1987, now abandoned.

BACKGROUND

The endothelium is a single layer of flattened transparent cells whichare joined edge to edge, with the result that they form a membrane ofcells. Endothelial cells originate during development from the embryonicmesoblast or mesoderm. They occur on the free surfaces of serousmembranes, in the anterior chamber of the eye and on the surface of thebrain and spinal cord. In addition, they form the lining membrane of theheart, blood vessels, and lymphatics.

It is possible, using methods developed in recent years, to attaininterspecies genetic recombination. Genes derived from differentbiological classes are able to replicate and be expressed in a selectedmicroorganism. Therefore, it is possible to introduce into amicroorganism genes specifying a metabolic or synthetic function (e.g.,hormone synthesis, protein synthesis, nitrogen fixation) which ischaracteristic of other classes of organisms by linking the genes to aparticular viral or plasmid replicon.

Since the late 1970s, progress has been made toward the development ofgeneral methods for introducing cloned DNA sequences into mammaliancells. At the present time, however, there is a need for an effectivemethod of stably introducing selected genetic material of interest intoendothelial cells and enabling them to express it, thus producing theencoded protein or polypeptide.

SUMMARY OF THE INVENTION

The invention described herein relates to genetically engineeredendothelial cells and particularly to genetically engineered endothelialcells which express selected genetic material of interest (DNA or RNA)which has been incorporated into them, for example by means of aretroviral vector having a recombinant genome which includes the geneticmaterial of interest. It also relates to methods of stably introducinginto endothelial cells such genetic material and methods of using thegenetically engineered endothelial cells.

Endothelial cells of this invention have stably incorporated in themgenetic material of interest, which encodes a product (e.g., a protein,polypeptide, or functional RNA) whose production in the endothelialcells is desired. The modified endothelial cells express theincorporated genetic material (produce the encoded product). Thisgenetic material of interest is referred to herein as incorporatedgenetic material. The incorporated genetic material can be any selectedDNA of interest (e.g., all or a portion of a gene encoding a product ofinterest) or RNA of interest. It can be, for example, DNA or RNA whichis present in and expressed by normal endothelial cells; DNA or RNAwhich does not normally occur in endothelial cells; DNA or RNA whichnormally occurs in endothelial cells but is not expressed in them atlevels which are biologically significant (i.e., levels sufficient toproduce the normal physiological effects of the protein or thepolypeptide it encodes); DNA or RNA which occurs in endothelial cellsand has been modified so that it is expressed in endothelial cells; andany DNA or RNA which can be modified to be expressed in endothelialcells, alone or in any combination thereof. Endothelial cells of thepresent invention can also express genetic material encoding aselectable marker, thus providing a means by which cells expressing theincorporated genetic material are identified and selected for in vitro.Endothelial cells containing incorporated genetic material are referredto as transduced endothelial cells.

In particular, retroviral vectors have been used to stably transduceendothelial cells with genetic material which includes genetic materialencoding a polypeptide or protein of interest not normally expressed atbiologically significant levels in endothelial cells. The geneticmaterial introduced in this manner can also include genetic materialencoding a dominant selectable marker. Genetic material including DNAencoding a polypeptide of interest alone or DNA encoding a polypeptideof interest and a dominant selectable marker has been introduced intocultured endothelial cells. Expression of these genes by the endothelialcells into which they have been incorporated (i.e., endothelial cellstransduced by the use of retroviral vectors) has also been demonstrated.

Because genes can be introduced into endothelial cells using aretroviral vector, they can be "on" (subject to) the retroviral vectorcontrol; in such a case, the gene of interest is transcribed from aretroviral promoter. Alternatively, retroviral vectors having additionalpromoter elements (in addition to the promoter incorporated in therecombinant retrovirus) which are responsible for the transcription ofthe genetic material of interest, can be used. For example, a constructin which there is an additional promoter modulated by an external factoror cue can be used, making it possible to control the level ofpolypeptide being produced by the endothelial cells by activating thatexternal factor or cue. For example, heat shock proteins are proteinsencoded by genes in which the promoter is regulated by temperature. Thepromoter of the gene which encodes the metal-containing proteinmetallothionine is responsive to cadmium (Cd++) ions. Incorporation ofthis promoter or another promoter influenced by external cues also makesit possible to regulate the production of the polypeptide by theengineered endothelial cells.

Endothelial cells may be transduced in two general settings in vitro orin vivo. Both settings require the use of a method for the transfer ofgenetic material of interest into endothelial cells, such as through useof a recombinant retroviral vector or other vector. For in vitrotransduction, endothelial cells grown in tissue culture vessels areexposed to a vector, such as a recombinant retrovirus encoding thegenetic material of interest, thereby producing transduced endothelialcells. Endothelial cells transduced in vitro with the genetic materialare then transplanted using one of a variety of known methods. Suchmethods include, but are not limited to, the transplantation ofsynthetic vessels or prosthetic valves lined with transduced endothelialcells or the transplantation of a device or matrix designed to housetransduced endothelial cells.

Alternatively, the transduction can be performed in vivo by applying themethod for transfer of genetic material of interest to endothelial cellsin a tissue or organ. For in vivo transduction, endothelial cellspresent in a tissue or organ are exposed, for example, to a recombinantretrovirus encoding the genetic material of interest. Such methodsinclude, but are not limited to, the site directed administration ofrecombinant retrovirus into a specific organ, limb, or blood vessel(e.g., via a catheter). Unlike endothelial cells transduced in vitro,these endothelial cells transduced in vivo would not require methods fortheir subsequent transplantation.

A method of transplanting endothelial cells transduced in vitro is alsoa subject of the present invention. Endothelial cells which have beentransduced in vitro are particularly useful for improving prostheticimplants (e.g., vessels made of synthetic materials such as Dacron andGortex.) which are used in vascular reconstructive surgery. For example,prosthetic arterial grafts are often used to replace diseased arterieswhich perfuse vital organs or limbs. However, the currently availablegrafts are usually made of synthetic material and are subject to manycomplications, the worst of which is a high rate of re-stenosis orocclusion. Animal studies suggest that lining the graft with autologousendothelial cells prior to implantation may decrease, but not prevent,graft reocclusion with its attendant morbid consequences.

However, endothelial cells can be modified according to the method ofthe present invention in a way that improves their performance in thecontext of an implanted graft. Examples include secretion or expressionof a thrombolytic agent to prevent intraluminal clot formation,secretion of an inhibitor of smooth muscle proliferation to preventluminal stenosis due to smooth muscle hypertrophy, and expression and/orsecretion of an endothelial cell mitogen or autocrine factor tostimulate endothelial cell proliferation and improve the extent orduration of the endothelial cell lining of the graft lumen.

For a similar application, endothelial cells of the present inventioncan also be used to cover the surface of prosthetic heart valves todecrease the risk of the formation of emboli by making the valve surfaceless thrombogenic.

Endothelial cells transduced by the method of the subject invention or avascular implant lined with transduced endothelial cells can also beused to provide constitutive synthesis and delivery of polypeptides orproteins, which are useful in prevention or treatment of disease. Inthis way, the polypeptide is secreted directly into the bloodstream ofthe individual. Currently available methods, in contrast, involveparenteral administration of the desired polypeptide.

In addition, there is no need for extensive (and often costly)purification of the polypeptide before it is administered to anindividual, as is generally necessary with an isolated polypeptide(e.g., insulin). Endothelial cells modified according to the presentinvention produce the polypeptide hormone as it would normally beproduced.

Another advantage to the use of genetically engineered endothelial cellsis that one can target the delivery of therapeutic levels of a secretedproduct to a specific organ or limb. For example, a vascular implantlined with endothelial cells transduced in vitro can be grafted into aspecific organ or limb; or the endothelial cells of a particular limb,organ or vessel can be transduced in vivo. The secreted product of thetransduced endothelial cells will be delivered in high concentrations tothe perfused tissue, thereby achieving a desired effect to a targetedanatomical location. This product will then be diluted to nontherapeuticlevels in the venous circulation during its return to the heart.

Another important advantage of the delivery system of this invention isthat because it is a continuous delivery system, the short half lives ofhormone polypeptides is not a limitation. For example, the half life ofhuman growth hormone (HGH) is approximately 19 minutes and parathyroidhormone, approximately 21/2 to 5 minutes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of a wild type murine leukemiavirus (retroviral) genome.

FIGS. 2A-2D are schematic representations of retroviral vectors, eachhaving a recombinant genome, useful in the present invention. FIG. 2A ispLJ and FIG. 2B is pEm, FIG. 2C is MFG and FIG. 2D is α-SGC.

FIG. 3 is a schematic representation of the construction of arecombinant retroviral vector, using the pLJ vector represented in FIG.2A and the human parathyroid hormone gene.

FIG. 4A-4D includes photographs of bovine aortic endothelial cellsinfected with a retrovirus that expresses low density lipoproteinreceptor (LDLR) and analyzed for the uptake of fluorescent labeled LDL.Panel A--uninfected bovine aortic endothelial cells under phasecontrast; Panel B--uninfected bovine aortic endothelial cells underfluorescent illumination; Panel C--infected bovine aortic endothelialcells under phase contrast; Panel D--infected bovine aortic endothelialcells under fluorescent illumination.

FIGS. 5A and 5B, includes photographs of bovine aortic endothelial cellsinfected with a retrovirus that expresses beta-galactosidase andanalyzed for its expression using an in situ histochemical stain. PanelA--uninfected bovine aortic endothelial cells stained forbeta-galactosidase activity; Panel B infected unselected bovine aorticendothelial cells stained for beta-galactosidase activity.

FIG. 6 is a pictorial representation depicting the transplantation ofgenetically modified endothelial cells into dogs.

FIG. 7 is a schematic representation of the modification of the tPAgene, the oligonucleotides used to facilitate the modification and theinsertion of the modified tPA gene into the vector MFG described in FIG.2C.

FIG. 8 is a photograph of a culture of endothelial cells identified bythe expression of factor VIII-related antigen.

FIG. 9a is a photograph of canine endothelial cells infected with aretrovirus that expresses tPA. A dark cytoplasmic stain is seen in thosecells expressing tPA.

FIG. 9B is a photograph of control cells not infected with the tPAretrovirus.

FIG. 10 is a photograph of an autoradiograph of a Southern blot ofcellular genomic DNA showing the stable integration into endothelialcells of the MFG-tPA and α-SGC-tPA recombinant retroviruses.

FIG. 11 is a photograph of an autoradiograph of a Northern blot ofcellular RNAs showing the expression of RNAs from the MFG-tPA andα-SGC-tPA recombinant retroviruses.

FIG. 12 is a histogram showing the potency after implantation into dogsof synthetic grafts lined with endothelial cells genetically augmentedto express tPA.

FIG. 13A is a diagram of the factor VIII polypeptide.

FIG. 13B is a diagram of the factor VIII cDNA showing the restrictionenzyme sites used in the various constructs to generate the retroviralvector.

FIG. 13C is a diagram of the deletion derivative of the factor VIII cDNAinserted into the retroviral vector with the deleted region shown asvertical lines.

FIG. 13D is an expanded diagram of the B domain deletion between theHind III and Pst I sites. The nucleotide sequence at the junction of theheavy chain and light chain is denoted above the line and thecorresponding amino acid numbers are denoted below the line.

FIG. 14 is a diagram of the assembled final retroviral vector,MFG-factor VIII.

FIG. 15 is a photograph of an autoradiograph of a Southern blot ofcellular genomic DNA showing the stable integration into endothelialcells of the MFG-factor VIII retrovirus.

FIG. 16 is a diagram of the a-SGC-LacZ recombinant retrovirus.

FIG. 17A is a low magnification photograph of an artery transduced invivo with the α-SGC-LacZ retrovirus.

FIG. 17B is a high magnification photograph of the same.

FIG. 17C is a segment of nontransduced artery.

FIG. 18 is a map of retroviral vector α-SGC.

FIG. 19 is a map of the retroviral vector MFG.

DETAILED DESCRIPTION OF THE INVENTION

Genetic material of interest has been incorporated into endothelialcells and expressed in the resulting genetically engineered endothelialcells. Genetic material of interest incorporated into endothelial cellsaccording to the method described can be any selected DNA of interest(e.g., all or a portion of a gene encoding a product of interest) or anyselected gene encoding a product of interest) or any selected RNA ofinterest. For example, it can be DNA or RNA which is present andexpressed in normal endothelial cells; DNA or RNA which does notnormally occur in endothelial cells; DNA or RNA which normally occurs in2-endothelial cells but, is not expressed in them at levels which arebiologically significant (levels sufficient to produce the normalphysiological effect of the protein or polypeptide it encodes); DNA orRNA which occurs in endothelial cells and has been modified in such amanner that it can be expressed in such cells; and any DNA or RNA whichcan be modified to be expressed in endothelial cells, alone or in anycombination thereof. This genetic material of interest is referred toherein as incorporated genetic material.

Endothelial cells of the present invention express the incorporatedgenetic material. For example, the endothelial cells of the presentinvention express genetic material encoding a polypeptide or a proteinof interest (genetic material of interest). Endothelial cells of thepresent invention can also include and express a gene encoding aselectable marker. Endothelial cells which express incorporated geneticmaterial are referred to herein as transduced endothelial cells.

Genetic material of interest which is DNA normally present in andexpressed by endothelial cells can be incorporated into endothelialcells with the result that they are able to overproduce the desiredprotein, polypeptide, or RNA.

As described in detail herein, genetic material encoding a hormone hasbeen introduced into endothelial cells by exposing them to media thatcontains a virus having a recombinant genome (i.e., by infecting them).The media used was a vital supernatant obtained by harvesting media inwhich recombinant virus producing cells have been grown. That is,producer cells have been grown in tissue culture to a confluent densityin Dulbecco's Modified Eagle's medium (DME) with 10% calf serum (CS) andpenicillin and streptomycin. Fresh media was added and subsequently(e.g., approximately 12 hours later), the media was harvested.Approximately 10 ml of media was harvested from a 10 cm plate ofconfluent producer cells. The harvested media (or viral stock) wasfiltered through a 0.45 micron Millipore filter to remove detachedproducer cells and was used immediately to infect cells or is stored at-70 C. Media was removed from a subconfluent plate of endothelial cells(recipient endothelial cells) and quickly replaced with viral stock(e.g., 5 ml/10 cm. plate) containing 8 mcg/ml of polybrene (Aldrich).Subsequently (e.g., approximately 12 hours later), this was removed andreplaced with fresh media.

The recombinant genome of the infectious virus includes the geneticmaterial of interest, which is incorporated into endothelial cells. Therecombinant genome can also have genetic material encoding a dominantselectable marker. Transduced endothelial cells which express apolypeptide not normally expressed by such cells at biologicallysignificant levels and, optionally, a dominant selectable marker havebeen made, as described herein.

The recombinant genome in one instance included genetic materialencoding human parathyroid hormone (hPTH). In another instance, therecombinant genome also included a gene encoding a dominant selectablemarker (e.g., the neo gene which encodes neomycin resistance in bacteriaand G418 resistance in mammalian cells). As a result, the endothelialcells were transduced--that is, the genetic material of interest (inthis case, DNA encoding hPTH and, optionally, the neo gene) was stablyintroduced into the endothelial cells. The transduced endothelial cellsexpress the encoded hPTH alone or in addition to the neo resistanceprotein, resulting in cells having the selectable trait.

In another instance, the recombinant genome included only the geneticmaterial of interest (e.g. factor VIII clotting protein or tissueplasminogen activator (tPA) and not a gene encoding a dominantselectable marker (e.g. neo gene). As a result, the transducedendothelial cells express the factor VIII protein or tPA in the absenceof the dominant selectable marker gene.

As described below, endothelial cells have been transduced with geneswhich code for secreted products (e.g., human parathyroid hormone (hPTH)(see Example 1) tissue plasminogen activator (tPA) (see Example 5) andhuman clotting factor VIII protein (see Example 6); a gene which codesfor a membrane receptor (e.g., low density lipoprotein receptor (LDLR)(see Example 2); and a gene coding for an intracellular bacterial enzyme(e.g., beta-galactosidase) (see Example 3).

The transduction of endothelial cells may be performed either in vitroor in vivo. For example, in vivo transduction of endothelial cells maybe carried out using a recombinant retrovirus which is introduced intoan individual via site directed administration of the recombinantretrovirus into a specific organ, limb or blood vessel (e.g., via acatheter, as described in Example 8). The in vivo transduction ofendothelial cells has several advantages, one of which is the lack of aneed for a method to transplant the transduced endothelial cells.Endothelial cells transduced in vitro are first grown in tissue culturevessels, removed from the culture vessel, and introduced into orimplanted into the body.

Endothelial cells transduced in vitro can be introduced into therecipient by one of several methods. For example, transduced endothelialcells can be seeded onto the lumen of a prosthetic vessel, where theywill then grow to confluence, covering the lumen of the vessel. Thecells form the lining of what has been called a neointima, a wellorganized structure that resembles the intima and media of a nativevessel (i.e., a covering of endothelial cells with deeper layers ofsmooth muscle cells). The feasibility of this approach has beendemonstrated by experiments in which vascular grafts, seeded withautologous retrovirus-transduced endothelial cells were implanted intodogs (see Example 4).

External jugular veins harvested from adult mongrel dogs were used as asource of endothelial cells which were plated in vitro during a 10-14day period by two serial passages. Cells from each animal were dividedinto two aliquots and were either infected with a replication defectiveretrovirus containing the reporter gene, beta-galactosidase or were mockinfected. Small diameter dacron grafts were seeded at subconfluentdensities with endothelial cells by the autologous clot method andsurgically implanted as carotid interposition grafts into the dog fromwhich the cells were harvested; each dog received a graft seeded withthe genetically modified cells and a contralateral graft seeded withmock infected cells. Five weeks after implantation, the grafts wereharvested and analyzed.

Cells were enzymatically harvested from the luminal surface of a portionof the graft to permit a more detailed characterization. Primarycultures of cells were established and expanded in vitro forapproximately 2-3 weeks prior to analysis. Genetically modifiedendothelial cells were identified in this population by Southernanalysis and by the cytochemical assay for vector-expressedbetagalactosidase which is encoded by the lacZ gene.

The majority of cells (less than 95%) harvested from the graft andexpanded in vitro retained differentiated endothelial function. However,the proportion of cells that expressed viral directed beta-galactosidaseor contained proviral sequences was consistently decreased 2-10 foldwhen compared to the cultures that were analyzed at the time of seeding.This disparity is due in part to the partial repopulation of grafts withendogenous cells by growth through interstices or from the anastomoses.The transduced cells persisted on the lumen of the graft for at leastfive weeks and the transferred gene continued to function.

Alternatively, endothelial cells that have been transduced in vitro canbe grafted onto a blood vessel in vivo through the use of a catheter. Itis also possible to introduce transduced endothelial cells into bodycavities which are lined by serosal membranes, such as the peritonealcavity, the pleural space, and the pericardial space. In this case, theendothelial cells seed the serosal lining and secrete the product intothe cavity. The product is then be absorbed via the lymphatic system.

The isolation and maintenance of endothelial cells from capillaries andlarge vessels (e.g., arteries, veins) of many species of vertebrates hasbeen well described in the literature. For example, McGuire and Orkindescribe a simple procedure for culturing and passaging endothelialcells from large vessels of small animals. McGuire, R. W. and R. W.Orkin, Biotechniques, 5:546-554 (1987).

Frequently, calf aorta is the source of endothelial cells. A typicalprotocol for isolation of endothelial cells from a large artery isdescribed below. Sections of aortas freshly harvested are placed understerile conditions, in a solution containing collagenase (e.g., 0.5mg/ml) for 15-20 minutes at 37 C. The aortas are then rinsed twice withtissue culture medium (e.g., RPMI 1640) and the lumenal sheet ofendothelial cells is removed in complete medium (RPMI containing 15 mmHepes, pH 7.4, penicillin/streptomycin and 20% fetal calf serum) bygentle agitation, according to the method of Booyse et al. Booyse, F. M.et al., Thrombosis Diath. Haemorrh., 34:825-839 (1975). The cell patchesare transferred to tissue culture flasks in complete medium.

The cells divide and eventually cover the plate; when the plate isconfluent, the cells can be passaged using standard tissue culturetechniques. The purity of the cultures is assessed by uptake offluorescent labeled acetylated LDL which is specifically taken up byendothelial cells. If the cultures are not pure (i.e., contaminated withcells other than endothelial cells, such as smooth muscle cells orfibroblasts), endothelial cells are cloned by limiting dilution andexpanded to yield pure cultures. The life span of endothelial cells islimited in culture but varies markedly, depending on the anatomicalsource and donor animal. Bovine aortic endothelial cells have beenmaintained in culture for at least 15-20 passages.

Canine endothelial cells can be isolated from explanted segments of theexternal jugular vein, and human cells from segments of either theumbilical or saphenous veins. All cells can be freed from the vesselwall by published procedures involving collagenase treatments whichreproducibly yield pure cultures of human endothelial cells but whichcan produce mixed cultures of smooth muscle and endothelial cells fromcanine veins (Hunter, T. J. et al., Trans. Am. Soc. Artif. Intern.Organs, 29:177182 (1983); Watkins, M. T. et al., J. Surg. Res., 36:588-596, 1984)). To limit the potential for smooth muscle cellovergrowth, the canine endothelial cells can be cultured inplasma-derived serum, a media supplement low in smooth muscle cellmitogens. All cultures can be monitored by immunohistochemicalprocedures which identify smooth muscle cells with a monoclonal antibodyrecognizing muscle-specific actin isoforms, and endothelial cells withan antisera which recognizes Factor VIII-related antigen (Wagner, D. D.,et al., J. Cell Biol. 95: 355-360, 1982), as well as labeled acetylatedLDL as discussed above.

Retroviral Vectors

Retroviruses are RNA viruses; that is, the viral genome is RNA. Thisgenomic RNA is, however, reverse transcribed into a DNA copy which isintegrated stably and efficiently into the chromosomal DNA of transducedcells. This stably integrated DNA copy is referred to as a provirus andis inherited by daughter cells as any other gene. As shown in FIG. 1,the wild type retroviral genome and the proviral DNA have three genes:the ˜gag, the pol and the env, which are flanked by two long terminalrepeat (LTR) sequences. The gag gene encodes the internal structural(nucleocapsid) proteins; the pol gene encodes the RNA directed DNApolymerase (reverse transcriptase); and the env gene encodes viralenvelope glycoproteins. The 5' and 3' LTRs serve to promotetranscription and polyadenylation of virion RNAs.

Adjacent to the 5' LTR are sequences necessary for reverse transcriptionof the genome (the tRNA primer binding site) and for efficientencapsidation of viral RNA into particles (the Psi site). Mulligan, R.C., In: Experimental Manipulation of Gene Expression, M. Inouye (ed),155-173 (1983); Mann, R., et al., Cell, 33:153-159 (1983); Cone, R. D.and R. C. Mulligan, Proceedings of the National Academy of Sciences,U.S.A., 81:6349-6353 (1984).

If the sequences necessary for encapsidation (or packaging of retroviralRNA into infectious virions) are missing from the viral genome, theresult is a cis acting defect which prevents encapsidation of genomicRNA. However, the resulting mutant is still capable of directing thesynthesis of all virion proteins. Mulligan and coworkers have describedretroviral genomes from which these Psi sequences have been deleted, aswell as cell lines containing the mutant genome stably integrated intothe chromosome. Mulligan, R. C., In: Experimental Manipulation of GeneExpression, M. Inouye (ed), 155-173 (1983); Mann, R., et al., Cell,33:153-159 (1983); Cone, R. D. and R. C. Mulligan, Proceedings of theNational Academy of Sciences, U.S.A., 81:6349-6353 (1984). The teachingsof these publications are incorporated herein by reference.

As described by Mulligan and coworkers, the Psi 2 cell line wasconstructed in the following manner: A mutant murine leukemia virus(MuLV) genome in which the region or the genome implicated in theencapsidation of viral RNA into virions is deleted (the Psi sequence inFIG. 1) was constructed. This genome was stably introduced in NIH3T3cells by DNA cotransfection and stable transfectants that produced allof the viral proteins used for encapsidation, yet budded noninfectiousparticles, were isolated. The mutant of MuLV was constructed by deleting351 nucleotides from an infectious proviral DNA clone between theputative env mRNA 5' splice site and the AUG that initiates the codingsequence for Pr 65 gag. The deletion was made from a Bal I site to a Pst1 site and a HindIII site was generated at the point of deletion.

pMOV. (pMOVPsi) was constructed as follows: Three purified DNA fragmentswere ligated together to construct pMOV Psi-. The first was obtained bydigesting pMOV Psi+ with Xho I to completion, followed by partialdigestion with EcoRI. Chumakov, I. et al., Journal of Virology,42:1088-1098 (1982). The fragment extending from the Xho I site at 2.0 Uin MuLV, through the 3' LTR, 3' mouse flanking sequence, all of pBR322,and ending at the EcoRI site was purified from an agarose gel afterelectrophoretic separation. Vogelstein, B. and D. Gillespie, Proceedingsof the National Academy of Sciences, USA, 761:615-619 (1979). The secondfragment was obtained by digestion of pMOV Psi+ with Bal I to completionfollowed by purification of the fragment extending from the Bal I sitein pBR322 through 5' mouse flanking sequence and 5' LTR to the Bal Isite located at 0.7 U of MuLV. HindIII linkers (Collaborative Research)were then blunt-ligated to this fragment with T4 DNA ligase, and thefragment was digested with excess HindIII and EcoRI. The LTR-containingfragment was purified from an agarose gel after electrphoreticseparation. The third fragment present in the final ligation reactionwas obtained from pSV2gag/pol where the gag/pol region of MuLV had beensubcloned into pSV2. Mulligan, R. C. and P. Berg, Science, 209:1422-1427(1980). pSV2-gag/pol was digested to completion with Xho I and HindIIIand the fragment extending from the HindIII site (changed from the Pst Isite at 1.0 U of MuLV) to the Xho I site at 2.0 of MuLV was purifiedfrom an agarose gel following electrophoretic separation. These threeDNA fragments were then mixed in equimolar amounts at a total DNAconcentration of 50 ug/ml. in ligase buffer (50 mM Tris-HCl pH 7.8!, 10mM MgCl₂, 20 mM dithiothreitol, 1.0 mM ATP, 50 ug/ml. bovine serumalbumin) and incubated with T4 DNA ligase for 18 hr. at 15 C. E. coliHB101 was transfected with the ligated DNA, and ampicillin resistanttransfectants were obtained. The plasmid DNA obtained from a number oftransformants was screened for the desired structure by digestion withappropriate restriction endonucleases and electrophoresis throughagarose gels. Davis, R. W. et al., Methods in Enzymology, 65:404-411(1980).

Cell lines containing the Psi mutant stably integrated into thechromosome were made by cotransfection of pMOV-Psi and pSV2gpt, a SV40hybrid vector capable of XG PRT expression. Mulligan, R. C. and P. Berg,Science, 209:1422-1427 (1980). Cells from gpt+ colonies obtained in thisway were cloned and established into three lines: Psi-1, Psi-2, andPsi-3.

The Psi 2 cell line described by Mulligan and co-workers was created bytransfecting NIH 3T3 endothelial cells with pMOV-Psi, which is anecotropic Moloney murine leukemia virus (Mo-MuLV) clone. pMOVPsiexpresses all the viral gene products but lacks the Psi sequence, whichis necessary for encapsidation of the viral genome. pMOV-Psi-expressesan ecotropic viral envelope glycoprotein which recognizes a receptorpresent only on mouse (and closely related rodent) cells.

Another cell line is the Psi am line, which are Psi-2-like packagingcell lines. These Psi-am cell lines contain a modified pMOV-Psi-genome,in which the ecotropic envelope glycoprotein has been replaced withenvelope sequences derived from the amphotropic virus 4070A. Hartley, J.W. and W. P. Rowe, Journal of Virology, 19: 19-25 (1976). As a result,they are useful for production of recombinant virus with amphotropichost range. The retrovirus used to make the Psi am cell line has a verybroad mammalian host range (an amphotropic host range) and can be usedto infect human cells. If the recombinant genome has the Psi packagingsequence, the Psi-am cell line is capable of packaging recombinantretroviral genomes into infectious retroviral particles. Cone, R. andMulligan, R. C. Proceedings of the National Academy of Sciences, USA,81:6349-6353 (1984).

Two other packaging cell lines are known as Psi CRIP and Psi CRE. Thesecell lines have been shown to be useful to isolate clones that stablyproduce high titers of recombinant retroviruses with amphotropic andecotropic host ranges, respectively. These cell lines are described inDanos, O. and R. C. Mulligan, Proceedings of the National Academy ofSciences, USA, 85: 6460-6464 (1988) and in U.S. patent application Ser.No. 07/239,545 filed Sept. 1, 1988. The teachings of the reference andthe patent application are incorporated herein by reference. Psi CRIPand Psi CRE have been deposited at the American Type Culture Collection,Rockville, Md., under accession numbers CRL 9808 and CRL 9807,respectively, under the terms of the Budapest Treaty.

The wild type retroviral genome has been modified by Cone and Mulliganfor use as a vector capable of introducing new genes into cells. Asshown in FIGS. 2A-2D, the gag, the pol and the env genes have all beenremoved and a DNA segment encoding the neo gene has been inserted intheir place. The neo gene serves as a dominant selectable marker. Theretroviral sequence which remains part of the recombinant genomeincludes the LTRs, the tRNA binding site and the Psi packaging site.Cepko, C. et al., Cell, 37:1053-1062 (1984).

Additional vector constructions which have been used in producingtransduced endothelial cells of the present invention are represented inFIGS. 2A-2D and are described in detail below.

pLJ. The characteristics of this vector have been described in Korman,A. J. et al., Proceedings of the National Academy of Sciences, USA,84:2150 (1987). This vector is capable of expressing two genes: the geneof interest and a dominant selectable marker, such as the neo gene. Thegene of interest is cloned in direct orientation into a BamHI/SmaI/SalIcloning site just distal to the 5' LTR, while, the neo gene is placeddistal to an internal promoter (from SV40) which is farther 3' than isthe cloning site (is located 3' of the cloning site). Transcription fromPLJ is initiated at two sites: 1) the 5' LTR, which is responsible forexpression of the gene of interest and 2) the internal SV40 promoter,which is responsible for expression of the neo gene. The structure ofpLJ is represented in FIG. 2A.

Vector pLJ is represented in FIG. 2A. In pLJ, the genetic material ofinterest is inserted just following the 5' LTR. Expression of thisgenetic material is transcribed from the LTR and expression of the neogene is transcribed from an internal SV40 promoter.

pEM. In this simple vector, the entire coding sequence for gag, pol andenv of the wild type virus is replaced with the gene of interest, whichis the only gene expressed. The components of the pEm vector aredescribed below. The 5' flanking sequence, 5' LTR and 400 bp ofcontiguous sequence (up to the BamHI site) is from pZIP. The 3' flankingsequence and LTR are also from pZIP; however, the ClaI site 150 bpupstream from the 3' LTR has been ligated with synthetic BamHI linkersand forms the other half of the BamHI cloning site present in thevector. The HindIII/EcoRl fragment of pBR322 forms the plasmid backbone.This vector is derived from sequences cloned from a strain of MoloneyMurine Leukemia virus. An analogous vector has been constructed fromsequences derived from the myeloproliferative sarcoma virus. Thestructure of pEm is represented in FIG. 2B.

Vectors without a selectable marker can also be used to transduceendothelial cells with genetic material of interest. Such vectors arebasically simplifications of the vectors previously described, in whichthere is such a marker. Vector pEm is represented in FIG. 2B; asrepresented, the main components of the vector are the 5' and 3' LTR,and the genetic material of interest, inserted between the two LTRs.

MFG

The MFG vector (ATCC accession no. 68754) is similar to the pEm vectorbut contains 1038 base pairs of the gag sequence from MMLV to increasethe encapsulation of recombinant genomes in the packaging cell lines,and 350 base-pairs derived from MOV-9 which contains the splice acceptorsequence and transcriptional start. An 18 base pair oligonucleotidecontaining NcoI and BamHI sites directly follows the MOV-9 sequence andallows for the convenient insertion of genes with compatible sites. TheMMLV LTR controls transcription and the resulting mRNA contains theauthentic 5' untranslated region of the native gag transcript followeddirectly by the open reading frame of the inserted gene. The structureof MFG is represented in FIG. 2C. A more detailed map of MFG is providedin FIG. 19.

MFG was constructed by ligating the 5' LTR containing XhoI/NdeI fragmentof the half-GAG retroviral vector (half-GAG is described in Bender, etal., J. Virol. 6I:1639-1646) to an XhoI/BamHI H4 histone promoterfragment. Retroviral vector pEMB was digested with NdeI and BamHI, andthe 3' LTR containing fragment was ligated to the half GAG fragmentalready ligated to the H4 fragment so as to produce an intermediateretrovirus vector containing 2 LTRs in the proper orientation and alsocontaining the H4 fragment within the viral portion of the vector. Theintermediate vector was then linearized by digestion with NdeI and theNdeI site in the pB322 portion of the vector was filled in by polymeraseand destroyed by ligation. The vector was subsequently digested withXhoI and the XhoI site was joined to an NdeI linker. The vector wassubsequently cleaved with BamHI and the large fragment containing bothLTRs and the pBR322 sequence) was purified.

A linker having XhoI and BamHI and having the following sequence:

    CTAGACTGCCATGGCGCG

    TGACGGTACCGCGCCTAG

was synthesized and ligated to both the BamHI site on the clearedintermediate vector and an NdeI/XbaI fragment from pMOV9 containing asplice acceptor site next to the NdeI edge! so as to form a circularvector, MFG as illustrated in FIGS. 2C and 19.

αSGC

The aSGC vector (ATCC accession number 68755) utilizes transcriptionalpromoter sequences from the α-globin gene to regulate expression of thetPA gene. The 600 base pair fragment containing the promoter elementadditionally contains the sequences for the transcriptional initiationand 5' untranslated region of the authentic α-globin mRNA. A 360 basepair fragment which includes the transcriptional enhancer fromcytomeglovirus precedes the α-globin promoter and is used to enhancetranscription from this element. Additionally, the MMLV enhancer isdeleted from the 3' LTR. This deletion is transferred to the 5' LTR uponinfection and essentially inactivates the transcriptional activatingactivity of the element. The structure of α-SGC is represented in FIG.2D. A more detailed description of α-SGC is provided in FIG. 18.

Introduction of Genetic Material into Endothelial Cells and Assessmentof Expression of the Genetic Material

The recombinant amphotropic retrovirus produced by the packaging cellline is used to infect endothelial cells. As described above, therecombinant genome of the amphotropic retrovirus can include a varietyof components, but in general is comprised of two LTRs and, in place ofthe gag, the pol and the env sequences, a second promoter sequence. Insome cases, it also includes a gene encoding a selectable marker (e.g.,neo).

Viral stocks, to be used in introducing genetic material of interestinto endothelial cells, are harvested, as described above, supplementedwith 8 micrograms per mil. (mcg/ml.) of polybrene (Aldrich) and added tothe culture of endothelial cells. If the titer of the virus is high(e.g., approximately 10⁶ Cfu per ml.), then virtually all endothelialcells will be infected and no selection (e.g., of endothelial cells intowhich the vector, including the recombinant genome, has been introduced)is required. If the titer is very low, then it is necessary to use aretroviral vector that has a selectable marker, such as neo. If aselectable marker is used, after exposure to the virus, the cells aregrown to confluence and split into selective media (e.g., mediacontaining the antiobiotic, G418).

The neo gene is a bacterial gene derived from the transposon Tn5, whichencodes neomycin resistance in bacteria and resistance to the antibioticG418 in mammalian cells. This neo gene acts as a dominant selectablemarker; its presence in a mammalian cell converts the cell into onewhich will grow in the presence of G418, an antibiotic which generallycauses cell death. As a result, the presence of this gene in a mammaliancell can be determined by culturing cells in media which contains G418.The recombinant retrovirus having this recombinant genome is referred toas the neo virus.

The recombinant retroviral vectors having the neo gene also have acloning site. As a result, genetic material of interest can beintroduced into the vector, incorporated into endothelial cells alongwith the neo gene and expressed by endothelial cells transduced with therecombinant retrovirus (referred to as endothelial cells havingincorporated genetic material).

It should be possible to express virtually any gene of interest inendothelial cells by means of a retroviral vector. Retroviral vectorsthat express genes that encode three different classes of proteins havebeen constructed: a secreted hormone or polypeptide (e.g., hPTH, tPA orfactor VIII), a membrane receptor (receptor for LDL, LDLR), and anintracellular enzyme (beta-galactosidase). Efficient expression of therecombinant retroviral vector when incorporated into endothelial cellshas been demonstrated and is described in detail in the examples.

Introduction of Genetic Material Encoding Other Proteins or Polypeptides

Genes encoding other proteins or polypeptides can also be introducedinto endothelial cells by means of an appropriate retroviral vector. Forexample, a gene encoding human growth hormone (hGH), a gene encodingclotting Factor IX or a gene encoding insulin can be introduced intoendothelial cells. Such genes can be introduced into endothelial cells,alone or in combination with a gene encoding a selectable marker, suchas the neogene.

These genes, as well as others can be introduced into endothelial cellsin the same manner as described above for the hPTH gene and theresulting transduced endothelial cells can be implanted into or appliedonto an appropriate site in the body.

Other Vehicles and Means for the Introduction of Genetic Material ofInterest into Endothelial Cells

It is also possible to use vehicles other than retroviruses togenetically engineer or modify endothelial cells. Genetic information ofinterest can be introduced into endothelial cells by means of any viruswhich can express the genetic material of interest in such cells. Forexample, SV40, herpes virus, adenovirus and human papilloma virus can beused for this purpose.

It is also possible to introduce genetic material of interest intoendothelial cells in such a manner that it is not incorporated stablyinto the recipient cells, but is expressed episomally (remains distinctor separate from the recipient cell genome).

In addition chemical or physical means can be used to introduce geneticmaterial of interest into endothelial cells. An example of a chemicalmeans is the commonly used calcium phosphate transfection procedure andan example of a physical means is electroporation whereby cells areexposed to an electric current which enables the entry into the cell ofgenetic material of interest.

Uses of Endothelial Cells Having Incorporated Genetic Material

Improvement of Performance of Vascular Grafts or Implants

Many important disease states involve stenosis or occlusion of arteriesthat supply vital organs. The pathologic mechanism most commonlyimplicated in such disease states is atherosclerosis. Examples includeangina pectoris and myocardial infarction due to coronary arterydisease; transient ischemic attacks and strokes due to cerebral vasculardisease; renal vascular hypertension, and ultimately, renal failure dueto renal artery stenosis; and claudication of the lower extremities,which is caused by vascular disease of peripheral arteries and, in itsmost severe form, can result in amputation. Unless the agents thatpredispose an individual to atherosclerotic lesions are eliminated(e.g., hypertension, cigarette smoking) the natural history of thesedisease states is usually progression of the atherosclerotic lesions,resulting in permanent damage or death.

An accepted and widely used therapeutic approach to advancedatherosclerotic disease is to bypass the site of major stenosis ofocclusion with a prosthetic vessel made of synthetic material, such asDacron or Gortex. More than 350,000 vascular grafts are implanted eachyear. One major problem with this approach is that the prosthetic vesselis extremely thrombogenic (i.e., it has the propensity to developclots), which leads to a very high rate of restenosis. It has beenpossible to reduce this problem by seeding the lumen of the prostheticvessels with autologous endothelial cells; grafts lined with endothelialcells are presumably less thrombogenic. It is in this setting thatmodified endothelial cells would be particularly useful.

Endothelial cells can be genetically modified according to the method ofthe present invention to improve their performance in the context of anendothelial cell-lined prosthetic implant. Existing protocols for usingendothelial cell-lined prosthetic implants are complicated by severalsignificant technological problems, most of which can be overcomethrough the use of genetically engineered endothelial cells.

A problem with endothelialized implants is that the lumen of theprosthetic vessel undergoes progressive narrowing due to theproliferation of smooth muscle cells between the wall of the prosthesisand the luminal surface. One way in which this can be prevented is tointroduce into the endothelial cells a gene that secretes a productwhich inhibits the growth of smooth muscle cells. Many types ofautocrine:paracrine growth factors that control proliferation ofmesenchymal and/or epithelial cells have recently been identified andtheir genes cloned. Such genes can be introduced into endothelial cellsusing the method of the present invention. The resulting transducedendothelial cells produce the cell growth inhibitor.

A further technical problem of endothelialized implant protocols is thatthe binding (plating) efficiency of the endothelial cells to theprosthetic graft is relatively low. Previous attempts to improve thishave been directed at modifying the composition of the graft surface andhave been of limited success. Using the method of the present invention,it is possible to introduce into endothelial cells a gene which encodesa membrane receptor. The lumen of the prosthetic vessel can then becoated with the ligand for the receptor, thereby facilitating binding ofendothelial cells to the luminal surface through the membranereceptor/ligand interaction.

Genetically engineered endothelial cells of the present invention can beused to decrease the thrombogenicity of endothelial cell-linedprosthetic grafts. The mechanism of clot formation is believed to beplatelet adhesion followed by deposition of fibrin and propagation ofclot. This could be minimized or possibly eliminated by seeding thegrafts with genetically engineered endothelial cells that secrete athrombolytic agent (e.g., an agent which dissolves clots, such as tissueplasminogen activator (TPA) or streptokinase).

Use of Modified Endothelial Cells to Deliver a Product to a Limb orOrgan

This invention can also be used to introduce into endothelial cellsgenes that secrete factors which would be beneficial to the limb ororgan perfused by the artery containing the prosthesis. For example, acommon clinical problem is the presence of extensive narrowing of smallvessels distal to the site of the prosthetic vessel. This ischaracteristic of the vascular disease associated with diabetesmellitus. Revascularization of the larger vessels with implants leads toincomplete reconstitution of perfusion to the affected limb or organ.

A way of promoting vascular flow to a compromised organ or limb is tomaximally dilate all afferent vessels. Attempts to do this with oral orparenteral medicines have resulted in little therapeutic benefit,accompanied by many systemic side effects. This is caused, in part, bythe fact that the vasodilator is not targeted to the appropriate tissue.Endothelial cells engineered to secrete a potent vasodilator such asatrial naturetic factor is an alternative approach. In this application,transduced endothelial cells proximate to the affected organ or limb canbe implanted or generated by in vivo transduction thus resulting in theaffected organ or limb being perfused with arterial blood containing avery high concentration of a vasodilator. This results in an increase inthe overall vascular perfusion. However, the vasodilator is diluted tonon-pharmacologic levels upon return to the heart, thereby obviating themany systemic side effects of vasodilators that occur when administeredin a systemic nonselective manner.

Use of Modified Endothelial Cells as a Delivery System

The present invention makes it possible to genetically engineerendothelial cells in such a manner that they produce a selected proteinor polypeptide, such as selected polypeptides and proteins not normallyproduced in endothelial cells in biologically significant amounts, andsecrete them into the bloodstream or other area of the body (e.g., thecentral nervous system). The endothelial cells formed in this way canserve as a continuous drug delivery system to replace present regimens,which require periodic administration (by injection, infusion etc.) ofthe needed substance.

For example, it can be used to provide continuous delivery of insulin,which, at the present time, must be isolated from the pancreas,extensively purified and then injected into the body by those whoseinsulin production or utilization is impaired. In this way, insulin canbe introduced into the body via a continuous drug delivery system and,as a result, there would be no need for daily injections of insulin.

Genetically engineered endothelial cells can also be used for theproduction of clotting factors. Hemophiliacs lack a protein calledFactor VIII, which is involved in clotting. Factor VIII is nowadministered by injection. However, transduced endothelial cells havinggenes encoding Factor VIII can produce and deliver Factor VIII in viro.

Incorporation of genetic material of interest into endothelial cells canbe particularly valuable in the treatment of inherited disease and thetreatment of acquired disease. In the case of inherited diseases, thisapproach is used to provide genetically modified endothelial cells andother cells which can be used as a metabolic sink. That is, suchendothelial cells would serve to degrade a potentially toxic substancethat had accumulated to high levels in the patient. For example,transduced endothelial cells expressing the gene encoding adenosinedeaminase can be used in treating an inherited form of severe combinedimmunodeficiency caused by a deficiency in the enzyme adenosinedeaminase which results in the accumulation of toxic purine nucleosides.Endothelial cells of the present invention can also be used in thetreatment of genetic diseases in which a product (e.g., an enzyme orhormone) normally produced by the body is not produced or is made ininsufficient quantities. Here, endothelial cells transduced with a geneencoding the missing or inadequately produced substance can be used toproduce it in sufficient quantities. For example, this can be used inproducing alpha-1 anitrypsin the missing protein or defective protein inan inherited form of emphysema.

There are many acquired diseases for which treatment can be providedthrough use of genetically engineered endothelial cells (i.e.,endothelial cells transduced with genetic material of interest). Forexample, such cells can be used in treating anemia, which is commonlypresent in chronic disease and often associated with chronic renalfailure (e.g., in hemodialysis patients)

In this case, endothelial cells having incorporated in them a geneencoding erythropoietin would correct the anemia by secretingerthropoitin thus stimulating the bone marrow to increase erythropoiesis(i.e. production of red blood cells).

Transduced endothelial cells of the present invention can also be usedto administer a low systemic dose of tissue plasminogen activator as anactivator to prevent the formation of thrombi. In this case, endothelialcells having incorporated genetic material which encodes tPA wouldinhibit clotting in an individual in whom thrombus prevention isdesired. This would be useful, for example, as a prophylactic againstcommon disorders such as coronary artery disease, cerebrovasculardisease, peripheral vasaular occlusive disease, vein (e.g., superficial)thrombosis, such as seen in pulmonary emboli, or deep vein thrombosis.Endothelial cells which contain DNA encoding calcitonin can be used inthe treatment of Paget's Disease, a progressive, chronic disorder ofbone metabolism. Present treatment relies on subcutaneous administrationof calcitonin.

Endothelial cells engineered to produce and secrete interleukins (e.g.,IL-1, IL-2, IL-3) can be used in several contexts. For example, theresult of some of the therapies now used (e.g., chemotherapy) isinduction of neutropenia (the presence of abnormally low numbers ofneutrophils in the blood), often caused by direct suppression of thebone marrow. For example, use of virtually all the chemotherapeuticagents, as well as AZT, used in the treatment of (AIDS) Acquired ImmuneDeficiency Syndrome, results in neutropenia. This condition results innumerous life-threatening infections. In these cases, administration of,for example, IL-3 through implantation of endothelial cells whichcontain genetic material encoding IL-3 and thus express and secrete IL-3can be used to increase the neutrophil count. In addition, theadministration of thrombopoietin, which stimulates the production ofplatelets, can be used in the treatment of numerous conditions in whichplatelet count is low. In this case, endothelial cells transduced withthe gene for thrombopoietin can stimulate platelet production.

Another related application of endothelial cells having incorporatedgenetic material is in the treatment of AIDS. Interleukin 2 andInterleukin 3, which stimulate the immune system, are potentiallyvaluable in the treatment of AIDS. These molecules could be delivered byendothelial cells which have been genetically engineered to producethese two polypeptides (which are now administered by periodicinjection).

Another use of the present invention is in the treatment of enzymedefect diseases. In this case the product encoded by the gene introducedinto endothelial cells is not secreted (as are hormones); rather, it isan enzyme which remains inside the cell. There are numerous cases ofgenetic diseases in which an individual lacks a particular enzyme and isnot able to metabolize various amino acids or other metabolites. Thecorrect genes for these enzymes could be introduced via transducedendothelial cells. For example, there is a genetic disease in whichthose affected lack the enzyme adenosine deaminase. This enzyme isinvolved in the degradation of purines to uric acid. It might bepossible, using the present invention, to produce transduced endothelialcells, which express the missing enzyme at sufficiently high levels todetoxify the blood as it passes through the area in which the transducedcells are present in the body.

The present invention also has veterinary applications. Transducedendothelial cells can be used, for example, in delivering substancessuch as drugs and hormones to animals, which would otherwise be providedby being injected periodically (e.g., daily or less frequently). Use ofthe modified endothelial cells of the present invention has theadvantage that the presence of the modified cells within the animal willprovide quantities of the encoded protein on an ongoing basis, thuseliminating the need for daily/periodic administration of the substance.

The present invention will now be illustrated by the following examples,which are not intended to be limiting in any way.

EXAMPLE 1 Production of Human Parathyroid Hormone in TransducedEndothelial Cells

At the BamHI cloning site of pLJ, genetic material of interest can beinserted. The genetic material of interest can be DNA as describedabove.

In particular, a copy of the gene encoding human parathyroid hormone(hPTH) has been cloned into this site, (e.g., into pLJ) in the followingway: The pLJ plasmid was digested with BamHI and subsequently treatedwith the enzyme calf intestinal phosphatase. Following this, the linearvector was fractioned on agarose gel and purified, using glass beads. Inaddition, the BamHI fragment containing the human PTH gene was preparedfrom the plasmid described by Hendy et al., which contains a completecDNA of human PTH cloned into pBR322. Hendy, G. N., et al., Proc. Natl.Acad. Sci. USA, 78:7365-7369 (1981). See FIG. 3.

A sub fragment of the PTH cDNA, containing 17 bp of 5' untranslated, allcoding and 155 bp of 3' untranslated sequence, was isolated by digestingthe initial plasmid with DdeI and HinfI and isolating the 600bpfragment. The fragment was incubated with DNA polymerase in the presenceof deoxynucleoside in phosphates, to fill in the recessed ends. BamHIlinkers were ligated to the blunt ends with T4 DNA ligase. An authenticBamHI restriction fragment was generated by digesting this litigationmixture with BamHI. This was then subcloned into the BamHI site ofpBR322, which is the plasmid used as the source of hPTH in vectorconstruction.

Equal quantities of the pLJ linear backbone and the BamHI PTH fragmentwere added together, in the presence of T4 DNA ligase. The resultingmixture was maintained under conditions appropriate for ligation of thetwo fragments. The ligation mixture was used to transform bacterialHB101, which were then plated onto agar containing kanamycin. Maniatis,T. et al., Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory, p.p. 250-251, 504; Bolivar, F. and K. Backman, In: Methodsin Enzymology, R. Wu (ed.), Vol. 68, Academic Press, N.Y. (1979). Theresulting colonies were analyzed for the recombinant plasmid.

Parathyroid hormone is a polypeptide which has a role in the regulationof calcium in the body. Although the hPTH gene is present in humanendothelial cells, it is not expressed in those cells at biologicallysignificant levels. Endothelial cells capable of making a polypeptidehormone such as hPTH, or another substance not normally made by suchcells at biologically significant levels, can be engrafted onto orimplanted into an individual and serve as a continuous synthesis anddelivery system for the hormone, or other substance.

The Psi am cells producing the recombinant virus construct whichcontained the hPTH-encoding DNA and DNA encoding a selectable marker(such as the neo gene), were used to produce a viral stock, as describedabove. The viral stock was harvested; endothelial cells to be transducedwith the virus containing the hPTH gene were incubated with the stock.In this case, a selectable marker is used to identify and select fortransduced endothelial cells by culturing on media containing G418. Ifthe viral titer is sufficiently high, essentially all endothelial cellsare infected and selection, using a selectable marker and appropriatemedia, is not needed.

The ability of endothelial cells transduced with the recombinantretrovirus having the hPTH gene to express the hPTH gene has beenassessed in vitro as follows: The bovine aortic endothelial cells werederived from an explant from the aorta of a calf. The cells have alimited life span and are referred to as secondary cultures. Bovineaortic endothelial cells were infected with virus and not selected inneomycin, as described above. Transduced bovine aortic endothelial cellswere seeded onto 10 cm tissue culture dishes and grown to confluence.Fresh culture media (DME with 10% CS and penicillin and streptomycin)was then added; this point is referred to subsequently as time zero. Atthe end of 24 hours, the media was removed and the cells were assayedfor the production of human PTH.

The aliquots were analyzed for the presence of hPTH using aradioimmunoassay (Nichols) which measures intact hPTH. The technique isdescribed in AllegroTM Intact PTH/Immunoassay System for theQuantitative Determination of Human Intact Parathyroid Hormone in Serum,Nichols Institute Diagnostics, San Juan Capistrano, Calif. (36B-2170,Effective 7/86 Revised), the teachings of which are incorporated hereinby reference. The assay has a sensitivity of approximately onenanogram/milliliter serum (ng/ml) and was shown to be specific for humanPTH in that it does not cross react with calf PTH, the results of theexperiments are reported as the production of hPTH, as measured by RIA,over time. Results are shown in Table I.

                  TABLE I                                                         ______________________________________                                        Production of hPTH in Transduced BAE Cells                                    Cell                                                                          PTH Production                                                                ______________________________________                                        Control BAE*        <10 pg/106/24 h                                           Transduced BAE      6.3 ng/106/24 h                                           ______________________________________                                         *BAE = bovine aortic endothelial                                         

Endothelial cells from a bovine aorta transduced with DNA encoding hPTHaccording to the method of the present invention have been deposited atthe American Type Culture Collection (Rockville, Md.) under depositnumber CRL9601.

EXAMPLE 2 Production of Human LDL Receptor in Transduced EndothelialCells

The cDNA for human LDL receptor (LDLR) was inserted into the pEm vector.The cDNA for LDLR was prepared for insertion into the pEM as follows.LDLR cDNA was excised from the vector pTZ1 (obtained from Dr. Goldstein,University of Texas Health Science Center) by digestion with HindIII.The 2.6 kb HindIII fragment containing all of the LDLR coding sequencewas blunt ended with Klenow fragment and BclI oligonucleotide linkers(from NEB) were ligated to the cDNA with T4 DNA ligase. Finally, thisligation mixture was digested with an excess of the enzyme BclI and thefragment was fractionated on an agarose gel and purified. This wasinserted into the BamHI cloning site of pEm as follows. pEm was digestedwith BamHI and the linearized plasmid was digested with calf intestinalphosphatase. Equal quantities of the linkered LDLR insert and pEmbackbone were mixed and ligated with T4 DNA ligase. The ligation mixturewas used to transform HB101 and ampicillin resistant colonies wereanalyzed for the appropriate retroviral vector. The pEm-LDLR vector wastransfected into the Psi-am cell line and clones that produced highquantity of recombinant virus were identified. Viral stocks were used toinfect cultures of bovine aortic endothelial cells as described for PTH.

In vitro assessment of LDLR expression was carried out as follows:confluent plates of control or transduced bovine aortic endothelialcells were incubated with LDL that had been conjugated with afluorescent label (obtained from Biomedical Tech Inc., Stoughton, Mass.)at a concentration of 10 ug/ml for approximately 8 hours. Followingthis, the cells were washed with PBS and fixed in 0.5% gluteraldehyde inPBS. They were then visualized under a fluorescent microscope for theuptake of fluorescent labeled LDL (*LDL). The results of this experimentare presented in FIG. 4. Briefly, the level of endogenous LDLR is low,as evidenced by the relative lack of *LDL uptake in uninfected cultures(See 4B) However, in cultures infected with the LDLR virus,approximately 30% of all cells take up detectable quantities of *LDL,thereby documenting efficient expression of the exogenous LDLR (See 4D).

EXAMPLE 3 Production of Beta-galactosidase in Transduced EndothelialCells

The gene encoding beta-galactosidase from E. coli was inserted into pLJand this vector was transfected into the Psi-am cell line, resulting inproduction of high titer retroviral stocks. The construction of thisvector and isolation of the producer cell line has been described byPrice and coworkers. Price, J. et al., Proceedings of the NationalAcademy of Sciences, USA, 84: 156-160 (1987) Stocks of virus encodingthe beta-galatosidase gene were used to infect bovine aortic endothelialcells, as described earlier. The infected cultures were analyzed forbeta-galatosidase expression using an in situ histochemical stain. SeePrice et al, above. Cultures were analyzed before and after selection inG418. The retroviral vector used in this experiment expresses bothbeta-galactosidase and the neo gene which confers resistance to G418.The histochemical stain was performed as described by Price et al.Briefly, cell cultures were fixed in 0.5% gluteraldehyde in PBS for 5minutes, washed with PBS and exposed to the reaction mixture for atleast 12 hours. The reaction mixture contains a substrate forbeta-galatosidase which, when hydrolyzed, turns blue and precipitates inthe cell. As a result, any cell expressing the viral encodedbeta-galactosidase will turn blue. The results of this experiment arepresented in FIG. 5. No beta-galactosidase activity is detected incultures that were not exposed to virus (FIG. 5A); infected culturesdemonstrate beta-galactosidase activity in about 30% of the cells (FIG.5B). These transduced cells are selected for by incubating them in thepresence of G418.

EXAMPLE 4 Production of Beta-galactosidase in Transduced EndothelialCells on the surface of Vascular Grafts Transplanted In Vivo

A pictorial representation of a typical protocol for transduction andtransplantation of endothelial cells is shown in FIG. 6. Endothelialcells were enzymatically harvested from external jugular veins of adultmongrel dogs which weighed 20-25 kg. Cells from each animal were dividedinto 2 aliquots; one to be infected with a replication defectiveretrovirus (see below) and the other to be mock infected. Theenzymatically harvested cells were used to establish primary cultures.Endothelial cells were plated on to fibronectin coated flasks andmaintained in M199 Medium supplemented with 5% plasma derived equineserum, penicillin, streptomycin, heparin and ECGF during two serialpassages over a 10-14 day period.

During this time period, cells were exposed to fresh stocks of virussupplemented with polybrene (8 ug/m1) every 3 days (18 hr/exposure). Atthe end of the second passage, cells were harvested and aliquots wereanalyzed directly, cryopreserved, or used to seed 6 cm×4 mm knitteddacron TM drafts (CR BARD, Billerica, Mass.) according to a modificationof the 4-step method of Yates (0.75 ×10⁶ cells were added to theautologous blood during the second and third step). Animals wereanesthetized and 6 cm segments of both carotid arteries were replacedwith the seeded grafts as described. Each animal received an implantseeded with infected endothelial cells and a contralateral graft seededwith mock infected cells. Five weeks after implantation, the animalswere anesthetized and the grafts were harvested and analyzed.

Replication-defective retroviruses with amphotropic host range were usedto stably introduce a reporter gene into the genomic DNA of theendothelial cells. The lacZ gene was used as the reporter gene becauseits product of expression, beta-galactosidase, can be detected in situthrough the use of enzyme histochemical assays that stain the cell'scytoplasm blue. (Example 3). Efficiency of retroviral infection wasestimated by Southern analysis which detects proviral sequences and bythe cytochemical stain for viral-expressed beta-galactosidase whichdetects infected cells in situ.

There were two recombinant retroviruses used in these studies. The BAGvector has been described previously by Price et al., Proceedings of theNational Academy of Science, USA, 84: 156-160 (1987). The BAG virus,containing the Lac Z-gene, expresses beta-galactosidase from the 5' LTR(Long Terminal Repeat) and a selectable marker (neo) from an SV40derived promoter which confers resistance to kanamycin in prokaryotesand G418 in eukaryotes. Approximately 5-15% of the endothelial cellsexposed to the BAG virus were infected (summarized in Table II);cultivation of these cultures in media supplemented with theaminoglycoside G418 effectively selected for transduced cells.

The BAL vector was derived from the previously described BA-LDLR vectorexcept LDLR cDNA sequences were replaced with the coding sequences forthe E. coli beta-galactosidase gene. The higher titer BAL virus,containing the Lac Z-gene, expresses betagalactosidase from a promoterderived from the chicken beta-actin. Approximately 50% of the cellsexposed to the BAL virus were transduced as measured by Southernanalysis and by the in situ cytochemical stain for beta-galactosidase.

In Southern analysis, high molecular weight DNA was isolated and analiquot (10 ug) was digested with Kpn I, fractionated on a 1% agarosegel, transferred to Zetabind and probed with a 1200 bp ClaI/EcoRIfragment that had been labeled to a high specific activity according tothe method of Feinberg and Volt. Cytochemical characterization ofcultured endothelial cells was demonstrated by both phase contrast andfluorescent micrographs. Retrovirus-infected endothelial cells wereanalyzed at the time of seeding and after removal from the implantedgraft for uptake of DIL-AC-LDL and for expression of viral-directedbeta-galactosidase. Preseeded and post-implantation cells that had beeninoculated with DIL-AC-LDL were assessed using phase contrast andfluorescent micrographs. Preseeded and post-implanted cells were alsoanalyzed for the expression of viral directed beta-galactosidase.

At the time of seeding, the cultures were analyzed for endothelial cellspecific function (i.e., uptake of acetylated LDL, and the presence ofVon Willebrands factor) and for the presence of antigens specific forsmooth muscle cells. These analysis indicated that more than 98% of thecells from each isolate were functioning endothelial cells.

The experimental system was a modification of a previously described dogmodel that has been used to study graft repopulation. Dogs 1-3 receivedimplants that had been seeded with BAG-infected, unselected endothelialcells while dogs 4-7 initially received implants seeded withBAL-infected, unselected endothelial cells. While these experiments werein progress, endothelial cells from dogs 4-7 were also infected with theBAG virus and expanded in culture in the presence or absence of G418, anaminoglycoside that selects for BAG-transduced cells. After 5 weeks, thegrafts were explanted for analysis and new grafts were implanted in dogs4-7 (referred to as dogs 4' 7'). Each of these dogs received a graftseeded with endothelial cells that were infected with BAG virus andselected in G418 and a contralateral graft seeded with BAG-infectedunselected endothelial cells. The second set of grafts were againexplanted after 5 weeks.

Table II summarizes the results of these experiments. Animals 4' through7' represent the second experiment performed on animals 4 through 7 (seetext). BAG-U represents BAG-infected endothelial cells unselected withG418 for transduced cells. BAG-S represents BAG-infected endothelialcells selected with G418 for transduced cells. BAL representsBAL-infected endothelial cells. MOCK represents mock infected cells. Eis an indication of the efficiency of infection as measured by thebeta-galactosidase stain. Potency of the graft after 5 weeks isindicated with a (Y) yes or (N) no. Coverage of the graft is anindication of the per cent of the surface repopulated as measured byscanning electron microscopy.

                                      TABLE II                                    __________________________________________________________________________    Summary of Implantation Experiments                                           Experimental Graft     Control Graft                                               Infection    Explant                                                                            Infection  Explant                                     Animal+                                                                            Virus                                                                              E   Patent                                                                            Coverage                                                                           Virus                                                                              E Patent                                                                            Coverage                                    __________________________________________________________________________    1    BAG-U                                                                               15 Y   50-90                                                                              MOCK  0                                                                              Y   50-90                                       2    BAG-U                                                                               5  Y   50-90                                                                              MOCK  0                                                                              Y   50-90                                       3    BAG-U                                                                               5  Y   50-90                                                                              MOCK  0                                                                              Y   50-90                                       4    BAL  40-60                                                                             N   --   MOCK  0                                                                              N   --                                          5    BAL  40-60                                                                             Y   0    MOCK  0                                                                              Y   0                                           6    BAL  40-60                                                                             Y   50-90                                                                              MOCK  0                                                                              Y   50-90                                       7    BAL  40-60                                                                             Y   50-90                                                                              MOCK  0                                                                              Y   50-90                                       4'   BAG-S                                                                              100 Y   50-90                                                                              BAG-U                                                                              10                                                                              N   --                                          5'   BAG-S                                                                              100 Y   0    BAG-U                                                                              10                                                                              N   --                                          6'   BAG-S                                                                              100 Y   0    BAG-U                                                                              10                                                                              Y   50-90                                       7'   BAG-S                                                                              100 Y   50-90                                                                              BAG-U                                                                              10                                                                              Y   0                                           __________________________________________________________________________

Analysis of the explanted grafts revealed that 18 of 22 remained patentafter 5 weeks. Scanning electron microscopy demonstrated a lining ofcells with endothelial-like morphology on the luminal surface of 14 of18 patent grafts. When present, the endothelial cell lining wasincomplete (50-90% of the total surface) and patchy in distribution witha paucity of cells seen along the peaks of the crimped dacron graft. Aportion of each graft was fixed, stained for cells that expressviral-derived betagalactosidase and visualized through a high powerdissecting microscope with a magnification of 75OX. Each graft seededwith infected endothelial cells that remained patent and retainedluminal cells did contain beta-galactosidase-positive cells on the lumenof the vessel. Contralateral grafts seeded with MOCK infectedendothelial cells never demonstrate positive staining cells.

In situ analysis of the grafts was performed for viral-transduced cells.Longitudinal sections of the genetically engineered implants wereanalyzed for cells that express viral-expressed beta-galactosidase.Grafts were cut longitudinally and a portion fixed in 0.5%gluteraldehyde for 5 minutes, washed in PBS three times, and incubatedin x-gal solution for 2 hours and the luminal surface photographed witha Leitz dissecting microscope. The grafts were visualized enface andseveral interesting aspects of the seeding pattern were observed. Thedensity of transduced cells was greatest in the deeper surfaces of thecrimped graft. This was visualized under low power as rings of bluestaining cells that line the crevices of the serrated surface andcorrelates with the variation in surface endothelial cell repopulationvisualized by scanning electron microscopy. In addition there was noregional variation in the density or pattern of transduced cells withrespect to proximity to the distal or proximal anastomosis. Finally, ineach case the proportion of luminal cells which were positive forbeta-galactosidase seemed qualitatively lower than the preparation ofbeta-galactosidase positive cells that were seeded.

Cells were enzymatically harvested from the luminal surface of portionsof the grafts to permit a more detailed characterization. Primarycultures of cells were established and expanded in vitro forapproximately 2-3 weeks prior to analysis. Genetically modifiedendothelial cells were identified in this population of cells bySouthern analysis and by the cytochemical assay for viral-expressedbeta-galactosidase. The majority of cells harvested from the graft andexpanded in vitro retained differentiated endothelial function (lessthan 95%). However, the proportion of cells that expressed vitaldirected beta-galactosidase or contained provital sequences wasconsistently diminished when compared to the cultures analyzed at thetime of seeding. This disparity is due in part to the partialrepopulation of grafts with endogenous cells by growth throughinterstices or from the anastomoses.

EXAMPLE 5 Increased Expression of tPA by Genetically Modified Canine andHuman Endothelial Cells

The data presented in the Examples cited above indicate thatretroviral-mediated gene transfer can easily be applied to bovine,canine, and human endothelial cells. The data also indicates that itresults in the proper expression of intracellular and secreted proteins.The use of retroviral-mediated gene transfer for the expression of atherapeutically relevant protein is indicated in the following section.

Tissue plasminogen activator (tPA) is a protein normally secreted byendothelial cells that promotes fibrinolysis of blood clots. Recombinantretroviral vectors encoding human tPA were constructed and used totransduce canine endothelial cells in order to demonstrate the enhanceddelivery of a therapeutically relevant protein from transducedendothelial cells.

The modifications of the tPA gene for cloning into the recombinantretroviral vectors are shown in FIG. 7. The coding sequences of humanuterine tPA were contained within a Sal I DNA fragment of a pUC-basedplasmid obtained from Integrated Genetics Inc. Framingham, Mass. The SalI fragment was derived by placing Sal I linkers at the SFaN I site atbase pair 6 and the Bgl II site at base pair 2090 of the original cDNA.The coding sequences extends from base pair 13 to base pair 1699.

From this original clone a fragment that could be cloned directly intothe MFG and α-SCG vectors described in the body of this patent wasderived. The Sal I fragment was first converted to a Bam HI fragment bythe addition of synthetic Bam HI linkers and then digest with therestriction enzyme Bgl II to yield a 109 base pair Bam HI to Bgl IIfragment and a 1975 base pair Bgl II to Bam HI fragment. To recreate themissing 100 base pairs of tPA coding sequences and the translationalstart codon, two 104 base pair oligo nucleotides were chemicallysynthesized and annealed to create a fragment with an Nco I site at the5' end and a Bgl II site at the 3' end. This oligo nucleotide wasligated onto the Bgl II site of the partial 1975 base pair tPA gene tocreate a 2079 base pair tPA gene with the identical coding sequence ofthe original molecule, but which can be easily obtained as an Nco I toBam HI fragment. It was inserted directly into the MFG and a-SGC vectors(the resulting vectors were given ATCC accession numbers 68726 and68729, respectively). These manipulations were performed by standardmolecular biological techniques (Molecular Cloning-A laboratory Manual,T. Maniatis, E. F. Frisch, and J. Sambrook), and are diagrammed in FIG.7.

Cell lines producing recombinant virus encoding MFG-tPA and α-SGC-tPAwere made from the Psi crip packaging cell line of Danos and Mulligancapable of producing recombinant retrovirus of amphotrophic host rangeProc. Natl. Acad. Sci. U.S.A. 85:6460 (1988)!. 10 ug of the specifiedDNAs and 1 ug of the plasmid pSV2neo were co-precipitated andtransfected onto the packaging cells by standard calcium phosphatetransfection procedures. Stably transfected clones were isolated aftergrowth for 14 days in selective media containing 800 ug/ml G418. 24 hourculture supernatants were obtained from confluent monolayers ofindividual clones and used to infect NIH 3T3 cells. The culturesupernatants were removed after 24 hours exposure, and the 3T3 cellswere refed with normal media and allowed to grow for an additional 72hours. Fresh media was placed on these cells for 6 hours and thesesupernatants were assayed for human tPA with a commercially availableELISA specific for human tPA (Immunobind-5, American Diagnostica Inc.,New York, N.Y.) From this screen, clones of the packaging cell lineproducing either the MFG-tPA recombinant virus or the α-SGC-tPArecombinant virus were selected and designated MFG 68 and α-SGC 22,respectively.

Canine endothelial cells were isolated from 10 cm segments of theexternal jugular vein by collagenase digestion as described T. J.Hunter, S. P. Schmidt, W. V. Sharp, and (1983) Trans. Am. Soc. Artif.Intern. Organs 29:177!. The cells were propagated on fibronectin-coatedtissue culture dishes in M199 media containing 5% plasma-derived equineserum, 50 ug/ml endothelial cell growth factor, and 100 ug/ml heparin.Purity of the cell cultures was determined by immunohistochemical assayfor the presence of Von Willebrands Factor and the absence of smoothmuscle cell specific α-actin. The day before transduction, theendothelial cells were seeded at 5.5×10³ cells/cm² in medium withoutheparin. The following day, the endothelial cells were exposed for 24hours to supernatants containing recombinant virus derived from eachproducer cell line to which was added 8 ug/ml polybrene. The viralsupernatants were removed, the cells feed with normal media and growthwas allowed to proceed for an additional 48 hours before analysis.

High molecular weight genomic DNA and total RNA were isolated fromcultures of endothelial cells by standard techniques (MolecularCloning-A Laboratory Manual T. Manjarls, E. F. Fritsch, and J.Sambrook). The DNA and RNA were analyzed by hybridization analysis witha ³² P-labeled DNA probe prepared from the entire tPA cDNA fragment.Standard techniques were used for electrophoretic separation, filtertransfer, hybridization, washing, and ³² P-labeling (Molecular Cloning-ALaboratory Manual T. Maniatis, E. F. Fritsch, and J. Sambrook) Theproduction of human tPA in transduced canine endothelial cells wasdemonstrated with a species specific immunocytochemical stain.Transduced cells were fixed in 3% formaldehyde for 10 minutes at roomtemperature and then permeabilized in 0.1% Triton X-100 for 5 minutes.The fixed cell monolayer was then incubated sequentially with a murinemonoclonal antibody to human tPA, with an alkaline phophatase conjugatedgoat anti-mouse antibody, and finally with a color reagent specific foralkaline phophatase. This procedure specifically stains those cellsexpressing human tPA and can be visualized by conventional lightmicroscopy. In addition, tPA secretion from transduced cells wasdetermined from confluent cell monolayers. Fresh media was placed on thecells for 6 hours, removed and clarified by centrifugation, and theamount of human tPA determined with a commercially available ELISA(Immunobind-5, American Diagnostica).

The efficiency of the transduction process is shown byimmunocytochemical stain of a population of cells mock transduced ortransduced with MFG-tPA. As shown in FIG. 9, after a single exposure ofthe cells to a vital supernatant harvested from MFG 68, essentially allof the cells are synthesizing human tPA as opposed to none of the cellsin the control. This was achieved without selection of any type fortransduced cells.

An immunological assay was conducted to determine the amount of tPA thatwas being secreted from transduced cultures. As shown below, cellstransduced with recombinant virus from either MFG 68 or α-SGC 22secreted large amounts of human tPA. Under similar conditions, humanendothelial cells in culture typically secrete approximately 1 ng of tPAHanss, M., and D. Collen (1987) J. Lab. Clin. Med. 109: 97104!.

                  TABLE III                                                       ______________________________________                                        Cells                                                                         tPA/million                                                                   cells/6 hours        ng human                                                 ______________________________________                                        uninfected K9          EC    0.0                                              MFG 68     K9          EC    150.1                                            α-SGC 22                                                                           K9          EC    302.8                                            ______________________________________                                    

As a further confirmation that the endothelial cells had been transducedwith recombinant virus from MFG 68 and a-SGC 22, DNA and RNA wasisolated from transduced cells and analyzed by hybridization to aradiolabeled tPA gene. An autoradiogram of the DNA analysis is shown inFIG. 10. No hybridization was detected in the uninfected controls, butsingle hybridizing species of the appropriate molecular weight was seenin the cells infected with the two recombinant vectors. Thisdemonstrates that the genetic information has been transferred to thegenome of these transduced cells.

Hybridization analysis of total RNA isolated from these cells confirmsthe protein and DNA results and is shown in FIG. 11. Again nohybridization was detected in the control cells but in the RNA derivedfrom the transduced cells hybridizing bands of the appropriate sizes canbe seen. RNA from the MFG 68 and α-SGC 22 recombinant virus producingcells is also shown as controls.

EXAMPLE 6 in vivo Function of Transduced Canine Endothelial CellsTransplanted on the Surface of Vascular Grafts

Endothelial cells were enzymatically harvested from external jugularveins of adult female mongrel dogs that weighed 20-25 kg and cultured inthe laboratory and analyzed for purity as described in Example 5. Onehalf of the cells isolated from each animal were transduced by twoexposures to supernatants harvested tPa from the MFG 68 cell lineproducing the MFG-tPA recombinant virus as described in the previoussection. The other half were mock transduced. Growth curves conducted oneach population showed no difference in growth characteristics. ELISAmeasurements were made on culture supernatants derived from each batchof transduced cells to assure that tPA was being secreted from theaugmented cells. These cells were then propagated in the laboratory forapproximately one week to obtain sufficient numbers of cells.

For each animal from which cells had been isolated, two vascular graftsmade of expanded Teflon (W. L. Gore and Associates, Inc. Flagstaff,Ariz.) were seeded with cells. One graft was seeded with mock transducedcells, and the other with cells transduced to secrete high levels oftPA. Each graft, measuring 0.4 cm×14 cm, was precoated with 1.5 ug/cm²fibronectin (Sigma Chemical Corp., St. Louis Mo.), and then seeded with2,200,000 endothelial cells/cm. The grafts were then incubated for anadditional 72 hours in culture. Prior to implant the ends were cut offeach graft and checked to assure cell coverage.

The same dogs from which the cells had been harvested were anesthetizedand 10 cm segments of the seeded grafts were implanted as aorta-iliacbypasses. Each dog received two contralateral grafts; one seeded withcontrol cells and the other seeded with cells that had been transducedto secrete high levels of tPA. Following implantation the performance ofthe grafts was monitored daily with a B-mode scanner which locates thegraft with ultrasound and assesses blood flow through the graft byDoppler measurements (Accuson, Inc.). No drugs to reduce thrombusformation were administered to the animals.

The results of graft performance in 6 different animals is shown in FIG.12. The implant model described above is an extremely stringent one andleads to rapid graft failure by occlusive clot formation. Normal graftfunction is denoted by solid bar, and a graft which is failing but stillfunctioning by a striped bar. In the first animal, the control graft andthe graft lined with transduced cells secreting enhanced levels of tPA(experimental) failed due to clot formation 24 hours after implant. Inall of the other five animals, the graft lined with transduced cellssecreting enhanced levels of tPA functioned longer than the graft withcells which had only been mock transduced. This difference varied from24 hours to several months. These results demonstrate that a therapeuticeffect can be achieved in vivo with transduced endothelial cells.

EXAMPLE 7 Production of Human Factor VIII from Transduced EndothelialCells

Endothelial cells were genetically augmented to produce human factorVIII by transducing cells with a retroviral vector, MFG, containing amodified human factor VIII gene (ATCC accession no. 68726). The modifiedfactor VIII cDNA contains all of the coding sequences for the A1, A2,A3, C1, and C2 domains, however the B domain is deleted from amino acids743 to 1648. The removal of the B domain and the insertion of themodified factor VIII gene into the retroviral vector MFG is described indetail below and depicted in FIG. 13.

A full-length cDNA without the 5' and 3' untranslated sequences wasobtained in a plasmid vector inserted between the restriction sites NcoI (5') and Xho I (3'). For removal of the B domain, the factor VIII cDNAwas subcloned into a plasmid vector in 4 fragments spanning thesequences on both the 5' and 3' sides of the B domain. The firstfragment of the factor VIII cDNA was subcloned between the restrictionsites Sal I and Pst I in the plasmid vector pUC 9. The plasmid vectorwas cut with Sal I and Pst I and the 5' phosphates were removed usingcalf intestinal phosphatase. A 1591 base pair Xho I (nucleotide 7263) toNde I (nucleotide 5672) fragment, and a 359 base pair Nde I (nucleotide5672) to Pst I (nucleotide 5313) fragment from the full-length cDNA wereisolated and ligated with the Sal I/Pst I digested plasmid vector.

To remove the majority of the sequences encoding the B domain whichjoins amino acids 742 to 1649 in the same translational reading frame, 4oligonucleotides were synthesized with a 5' Hind III site and a 3' Pst Isite covering 168 base pairs. The oligonucleotides extend from the HindIII site at nucleotide 2427 which encodes amino acid 742 followed byamino acid 1649 which is the first amino acid of the activation peptideof the light chain through to the Pst I site at nucleotide 5313. Theplasmid vector pUC 9 was digested with the restriction enzymes Hind IIIand Pst I, and the 5' phosphates were removed using calf intestinalphosphatase. The oligonucleotides were synthesized as 4 separatestrands, kinased, annealed and ligated between the Hind III site and thePst I site of the plasmid vector.

The subcloned Hind III/Pst I oligonucleotide was juxtaposed to the PstI/Xho I fragments in a plasmid vector pUC F8. To generate this plasmid,a new polylinker was inserted into a pUC 9 plasmid backbone with the newpolylinker encoding the restriction enzyme sites 5' Sma I-Bam HI-XhoI-Pst I-Hind III-Asp 718-Nco I-Hpa I 3' used. The plasmid vector wasdigested with the restriction enzymes Bam HI and Hind III, and the 5'phosphates were removed with calf intestinal phosphatase. A partial PstI/Bam HI digest of the Pst I/Xho I subclone was used to isolate the 3'terminal factor VIII fragment, and a Pst I/Hind III digest of thesubcloned oligonucleotides was used to isolate the heavy and light chainjunction fragment. They were ligated into the plasmid vector pUC F8between the BamHI and Hind III sites.

This subclone containing the factor VIII sequences between nucleotides2427 and 7205 was digested with Asp 718 and Hind III, and the 5'phosphates were removed using calf intestinal phosphatase. A fragmentencoding factor VIII between the restriction enzyme sites Asp 718(nucleotide 1961) and Hind III (nucleotide 2427) was isolated andligated into the plasmid vector to generate a subclone (pF8 3' delta)containing the factor VIII sequences from nucleotide 1961 through to thetranslational stop codon at nucleotide 7205.

The construction of the retroviral vector containing the modified factorVIII gene was carried out by inserting the factor VIII gene between therestriction sites Nco I and Bam HI of the retroviral vector MFG. Thefactor VIII subclone pF8 3' delta was digested with Sma I and convertedto a BglII site using an oligonucleotide linker. An Asp 718/Bgl IIfragment was isolated from the 3' factor VIII subclone, and a 5' factorVIII fragment containing the ATG for initiation of translation wasisolated as an Nco I (nucleotide 151)/Asp 718 fragment (nucleotide1961). The retroviral vector MFG was digested with Nco I and Bam HI, andthe 5' phosphates were removed using calf intestinal phosphatase. Thefactor VIII fragments were ligated into the retroviral vector yieldingthe final factor VIII retroviral construct, see FIG. 14.

The cell line producing the retroviral particles was generated bytransfection of the retroviral vector MFG/factor VIII into equal numbersof ecotropic packaging cells Psi CRE and amphotropic packaging cells PsiCRIP as described by Bestwick et al. (Proc. Natl. Acad. Sci. U.S.A.1988. 85:5404-5408.). To monitor the extent of superinfection takingplace between the 2 host ranges of packaging cells, the production ofbiologically active factor VIII was measured using the Kabi DiagnosticaCoatest for Factor VIII, Helena Laboratories, Beaumont, Tex. and theproduction of vital RNA was measured by an RNA dot blot analysis. At 21days post transfection, the mixture of transfected packaging cells wasco-cultivated with the amphotropic packaging cell line Psi CRIP-HIS. ThePsi CRIP HIS packaging cell line is a variant of the previouslydescribed Psi CRIP packaging cell line. The Psi CRIP HIS packaging cellline is identical to the Psi CRIP packaging cell line except that theretroviral envelop gene was introduced into the cell by cotransfectionwith pSV2-HIS plasmid DNA, a different dominant selectable marker gene.The packaging cell lines were cultured at a 1:1 ratio for isolation of ahomogeneous amphotropic retroviral stock of transducing particles. Thesuperinfection of the amphotropic packaging cell line Psi CRIP HIS hasled to the generation of a stable cell line, HIS 19, which producesrecombinant retrovirus that efficiently transduce the modified humanfactor VIII gene. Antibiotic selection of the retroviral producing cellline was not required to isolate a cell line which produces high-titerrecombinant retrovirus. The genomic DNA of the cell line has beencharacterized by Southern blot hybridization analysis to determine thenumber of integrated copies of the retroviral vector present in theproducer cell line. The copy number in the retroviral producing cellline is approximately 0.5, therefore on average 50% of the Psi CRIP-HISpackaging cells contain a copy of the retroviral vector with themodified factor VIII gene. The retroviral vector and the modified factorVIII gene are intact without any deletions or rearrangements of the DNAin the packaging cell line. The copy number of the retroviral vectorremains constant with the continuous passage of the retroviral producingcell line. For obtaining the highest titer of recombinant retrovirus,HIS 19 was carried 3 passages in selective histidine minus mediafollowed by 4 passages in completed DMEM media. For the generation ofretroviral particles, HIS 19 was seeded at 5×10⁵ -1×10⁶ cells in a 10 cmcell culture dish. At 48 hours postseeding, approximately 70%confluency, fresh medium (DMEM+10% calf serum) was added to the platesfor collection 24 hours later as the source of recombinant retrovirusfor transduction.

The modified factor VIII gene was transduced into canine endothelialcells isolated from the jugular vein. The endothelial cells were seededat 3X105 5 cells per 10 cm. dish in complete M199 medium with 5%. plasmaderived serum (Equine), 100 ug/ml heparin, and 50 ug/ml endothelial cellgrowth factor for 4-6 hours. The cells were then incubated overnight inM199 medium with 5% plasma derived serum, and 100 ug/ml endothelial cellgrowth factor overnight without heparin which adversely affects theefficiency of the transduction process. Cells were exposed to the freshviral supernatant plus polybrene (8 ug/ml) for 24 hours. After removalof the viral supernatant, the cells were put into M199 medium with 5%plasma derived serum, 100 ug/ml endothelial cell growth factor to growto approximately 70-80% confluence. At that time, the medium was changedto M199 medium with 5% heat inactivated fetal bovine serum (heated at66* C. for 2 hours), and 50 ug/ml of ECGF. Following a 24 hr.incubation, the medium was collected and assayed for biological activefactor VIII by the Kabi Coatest.

With this retroviral producing cell line, between 50% and 75% of theendothelial cells were transduced as determined by Southern blotanalysis. The factor VIII gene can be transduced at this frequency witha single exposure to the recombinant retrovirus, and without antibioticselection of the transduced cells. The transduced endothelial cellscontain an intact copy of the recombinant retroviral genome and themodified factor VIII gene without any deletions or rearrangements asshown in FIG. 15. The rate of production of biologically active factorVIII from the genetically augmented endothelial cells was 400 ng/5×10⁶cells/24 hrs.

EXAMPLE 8

In Vivo Transduction of the Endothelium

Using standard stocks of recombinant retrovirus made as described in theprevious examples, we have obtained preliminary data demonstrating thein vivo transduction of endothelial cells. The approach is based on thepreviously published observation (Reidy, Mass., Schwartz SM. Lab Invest44:301-308, 1981) that a defined injury to an artery surface removes asmall strip of endothelial cells and this denuded area heals withinseventy-two hours by proliferation and ingrowth of new endothelial cellsfrom the edge of the defect. Cell division is a requirement foreffective transduction by recombinant retroviruses and the injury of theendothelium with a wire is one of potentially many methods to induceendothelial cell proliferation our method uses Reidy's technique ofdefined injury to induce endothelial cell proliferation, then exposesthe proliferating cells directly to supernatants containing recombinantretroviral vectors. Our initial experiments document the ability of thismethod to successfully transduce endothelial cells in situ, thuspotentially avoiding the necessity of tissue culture techniques for thesuccessful introduction of new genetic sequences.

This method requires two surgical procedures, the first procedureinjures the blood vessel surface (here described for the right iliacartery) and induces the proliferation of endothelial cells. The secondprocedure delivers recombinant retrovirus to the cells undergoingreplication on the vessel surface, while preventing the flow of bloodfrom the proximal arterial tree while the proliferating cells areexposed to retroviral particles. For simplicity of performance theprocedure is described for iliac arteries.

To demonstrate in vivo gene transfer, we used the marker gene conceptpublished in 1987 (Price J., Turner D., Cepko C. 1987 Proc. Natl. Acad.Sci. U.S.A 84:156-160.) (see Example 3) with an improved vector based onthe α-SGC vector (FIGS. 2d and 18). The lacZ gene encodingbeta-galactosidase was inserted into the α-SGC vector to generate theα-SGC-LacZ vector which is represented in FIG. 16. This recombinantconstruct was transfected into the Psi Crip packaging cell line and aclone of Psi Crip cells producing high titers of the α-SGC-LacZrecombinant retrovirus were isolated as described in Example 5. Stocksof the αSGC-LacZ recombinant retrovirus were used for in vivotransduction.

The experimental animals (,rabbit) were anesthetized(ketamine/xylazine), both groins were shaved and prepped, and theanimals positioned on an operating table. Through bilateral verticalgroin incisions the common, superficial, and profunda femoral arterieswere exposed. On the right (the side to be injured) small branches offthe common femoral artery were ligated to insure that outflow from theisolated arterial segment would only occur through the internal iliacartery. If necessary, the inguinal ligament was divided and the vesselfollowed into the retroperitoneum to assure complete control of all sidebranches. The right superficial femoral artery (SFA) was ligated with3-0 silk approximately 1.5 cm below the profunda take-off, control ofthe SFA was obtained at the SFA/profunda junction, and a transversearteriotomy created. A fine wire (the styler of a 20 gauge Intracath wasused), doubled upon itself to provide springiness to assure contact withthe vessel wall, was passed up the common femoral and iliac arteryretrograde to produce the defined injury described by Reidy et al. Thewire was removed, a 20 gauge angiocath was inserted in the arteriotomyand secured to the underlying muscle for immediate access at the nextsurgical procedure. The incisions were closed in layers and the animalsallowed to recover.

Twenty-four hours later a recombinant virus containing supernatantharvested from a trip producer of the αSGC-LAC-Z vector and supplementedwith polybrene to a final concentration of 8 ug/ml was used for in vivotransduction. The animals were again anesthetized and both incisionsreopened in a sterile environment. To obtain control of the right iliacvessels above the area that had been injured with no disturbance to thepreviously denuded right iliac vessel, a #3 Fogarty™ balloon embolectomycatheter was inserted through an arteriotomy in the left superficialfemoral artery, passed to the aortic bifurcation and the ballooninflated to interrupt blood flow. The right profunda femoris artery wasoccluded. The supernatant (10 ml) containing the recombinant retroviruswas introduced by hand injection through the angiocath previously placedin the right SFA. The supernatant flowed in a retrograde fashion fromthe right common femoral to the right external iliac and into the rightinternal iliac artery. By leaving the right internal iliac artery openoutflow for the supernatant was allowed and a full 10 ml of supernatantcould be instilled. In the experiments performed to date thesupernatants have been exposed to the vessel wall for periods of four toeight minutes. The catheters from the left and right sides were thenremoved, hemostasis obtained, and the incisions closed.

Ten to fourteen days later animals were anesthetized prior to sacrifice.After anesthesia and prior to exposure, patency was assessed by directpalpation of the distal vessel. The infra-renal aorta and inferior venacava were surgically exposed, cannulated, and the vessels of the lowerextremity flushed with heparinized Ringer's lactate (2 U/ml) atphysiologic pressure (90 mmhg.) A lethal dose of nembutal wasadministered and the arteries perfusionfixed in situ in 0.5%gluteraldehyde in 0.1M cacodylate for 10 minutes. The aorta and bothiliac arteries were excised in continuity and rinsed in phosphatebuffered saline (PBS) with 1 mM MgCl2. The vessels were then stained forlacZ activity by incubation in the x-gal substrate for 1-1.5 hours at37* C. When the reaction was complete, the x-gal solution was washedaway and replaced with PBS, photographed and shown in FIG. 17.

Two experiments have been completed with this protocol. Both experimentsdemonstrated successful in vivo transduction as shown by the in situexpression of the lacZ gene product in cells on the surface of theartery as visualized by the selective intense blue staining in acytoplasmic pattern (FIG. 17). FIG. 17A and B is a segment of theexternal illiac artery injured with a wire, exposed to αSGC-LacZrecombinant retrovirus, fixed and stained for latz activity andphotographed with low magnification (FIG. 17A) and high magnification(FIG. 17B). Note the line of intensely stained blue cells consistentwith the pattern of injury and proliferation described by Reidy et al.FIG. 17C is a photograph at low magnification of the same artery distalto the site of virus injection which has been identically fixed andstained. This area has only modest and diffuse background staining.

Biological Deposits

On Oct. 3, 1991, Applicants have deposited with the American TypeCulture Collection, Rockville, Md., U.S.A (ATCC) the plasmid MFG withthe factor VIII insertion, described herein ATCC accession no. 68726,plasmid MFG with the tPA insertion, described herein, given ATCCaccession no. 68727, the plasmid α-SGC, described herein, with thefactor VIII insertion, given ATTC ascession no. 68728, and plasmid α-SGCwith the tPA insertion, described herein, given ATCC accession no.68729. On Oct. 9, 1991, Applicants have deposited with the American TypeCulture Collection, Rockville, Md., U.S.A (ATCC) the plasmid MFG,described herein, given ATCC accession no. 68754, and plasmid α-SGC,described herein and given ATCC accession no. 68755. These deposits weremade under the provisions of the Budapest Treaty on the InternationalRecognition of the Deposit of Microorganisms for the purposes of patentprocedure and the Regulations thereunder (Budapest Treaty). This assuresmaintenance of a viable culture for 30 years from date of deposit. Theorganisms will be made available by ATCC under the terms of the BudapestTreaty, and subject to an agreement between Applicants and ATCC whichassures unrestricted availability upon issuance of the pertinent U.S.patent. Availability of the deposited strains is not to be construed asa license to practice the invention in contravention of the rightsgranted under the authority of any government in accordance with itspatent laws.

Equivalents

Those skilled in the art will recognize, or be able to ascertian usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described specifically herein. Suchequivalents are intended to be encompassed in the scope of the followingclaims.

We claim:
 1. Transduced endothelial cells which have been engineered toexpress stably maintained genetic material encoding a polypeptide orprotein product.
 2. Endothelial cells according to claim 1 wherein thegenetic material is DNA, and wherein the genetic material is selectedfrom the group consisting of: DNA that is present in and expressed bynormal endothelial cells; DNA that does not normally occur inendothelial cells; or normally occurs in endothelial cells but is notexpressed in them at levels which are biologically significant; and DNAthat can be modified so that it can be expressed in endothelial cells.3. Endothelial cells according to claim 1, wherein said genetic materialencodes a protein or polypeptide selected from the group consisting: (a)a hormone; (b) an enzyme; (c) a selectable marker; (d) a protein orpolypeptide which is secreted from the cells; and (e) a membranereceptor.
 4. Endothelial cells of claim 1 wherein said stably maintainedgenetic material has been introduced by transduction.
 5. Encothelialcells of claim 4 wherein said transduction is via a retrovirus having arecombinant genome comprised of; (a) genetic material encoding theproduct of interest, (b) the long terminal repeat sequences, tRNAbinding site and Psi packaging site derived from a retrovirus; and (c)at least one promoter of eukaryotic origin which is capable of beingmodulated by an external cue.
 6. Endothelial cells according to claim 1wherein said genetic material encodes a thrombolytic protein. 7.Endothelial cells according to claim 1 wherein said endothelial cellsproduce a confluent layer of endothelial cells lining the inner surfaceof a prosthetic vessel, and wherein said genetic material encodes anendothelial cell mitogen or an endothelial cell growth factor. 8.Endothelial cells according to claim 1 wherein said genetic materialencodes a membrane receptor which binds a ligand, and wherein saidligand may be attached to a prosthetic vessel so that binding of theendothelial cells to the surface of the prosthetic vessel is enhanced.9. Endothelial cells according to claim 1 wherein said genetic materialencodes a product which inhibits smooth muscle cell growth, forinhibiting the growth of smooth muscle cells in a synthetic vascularimplant.
 10. Transduced endothelial cells according to claim 1 whereinsaid expressing is in vitro.
 11. Transduced endothelial cells accordingto claim 10 wherein said genetic material of interest is selected fromthe group consisting of: DNA which is present in and expressed by normalendothelial cells; DNA which does not normally occur in endothelialcells; DNA which normally occurs in endothelial cells but is notexpressed in them at levels which are biologically significant; and anyDNA which can be modified so that it can be expressed in endothelialcells.
 12. Transduced endothelial cells according to claim 11, furthercomprising DNA which encodes at least one selectable marker. 13.Transduced endothelial cells according to claim 11, wherein said geneticmaterial of interest encodes a protein or polypeptide selected from thegroup consisting of: a hormone, a receptor, an enzyme and a polypeptide.14. Transduced endothelial cells according to claim 13, wherein saidgenetic material of interest encodes a polypeptide or protein productselected from the group consisting of human parathyroid hormone, tissueplasminogen activator, Factor VIII, low density lipoprotein receptor orbeta-galactosidase.
 15. Transduced human endothelial cells that havebeen engineered to express recombinant genetic material of interestencoding a polypeptide or protein in vivo.
 16. Transduced humanendothelial cells according to claim 15, wherein said genetic materialof interest is selected from the group consisting of: genetic materialwhich is present in and expressed by normal endothelial cells; geneticmaterial which does not normally occur in endothelial cells; geneticmaterial which normally occurs in endothelial cells but is not expressedin them at levels which are biologically significant; and any geneticmaterial which can be modified so that it can be expressed inendothelial cells.
 17. Transduced human endothelial cells of claim 16,further comprising DNA which encodes at least one dominant selectablemarker.
 18. Transduced human endothelial cells according to claim 16 inwhich said genetic material of interest encodes a protein or polypeptideselected from the group consisting of: a hormone, a receptor, an enzymeand a polypeptide.
 19. Transduced human endothelial cells according toclaim 15 wherein said genetic material of interest encodes humanparathyroid hormone, tissue plasminogen activator, Factor VIII, lowdensity lipoprotein receptor, or beta-galactosidase.
 20. Culturedendothelial cells containing DNA that is integrated into said culturedendothelial cells, wherein said DNA encodes a protein not made bynaturally-occurring endothelial cells and a selectable marker. 21.Transduced endothelial cells according to claim 5 having geneticmaterial of interest integrated therein, wherein said genetic materialof interest was reverse transcribed from RNA which was provided as partof a retroviral vector, and wherein said transduced endothelial cellshave been engineered to express said incorporated genetic material ofinterest.
 22. Transduced endothelial cells according to claim 21,wherein said cells are human cells.
 23. A method of making transducedendothelial cells which express integrated genetic material of interestencoding at least one polypeptide of interest, comprising the stepsof:a) contacting endothelial cells with media containing an infectiousrecombinant retrovirus having a recombinant genome comprising thegenetic material of interest; and b) maintaining the endothelial cellsand the media containing the infectious recombinant retrovirus underconditions appropriate for infection of the endothelial cells by saidrecombinant retrovirus, thereby producing transduced endothelial cells.24. A method according to claim 23 wherein said infection of theendothelial cells occurs in vitro.
 25. A method of producing a confluentlayer of endothelial cells lining the inner surface of a syntheticprosthetic vessel, comprising applying to the inner surface of thevessel endothelial cells comprising incorporated genetic materialencoding an endothelial cell mitogen, under conditions appropriate forendothelial cell proliferation.
 26. A method according to claim 25,wherein the genetic material encodes endothelial cell growth factor. 27.A method of enhancing binding of endothelial cells to a surface of asynthetic vessel, comprising the steps of:a) applying to the surface ofsaid vessel a ligand, to produce a ligand-bearing surface; b) seedingonto said ligand-bearing surface, said endothelial cells wherein saidendothelial cells contain integrated genetic material encoding amembrane receptor which binds the ligand; and c) maintaining the vesselunder conditions appropriate for binding of said ligand and saidmembrane receptor.
 28. A method of producing a prosthetic vessel havingendothelial cells which express incorporated genetic material ofinterest on the inner surface of the vessel, comprising lining the innersurface of the vessels with endothelial cells produced by the method ofclaim
 24. 29. A method of producing a prosthetic vessel lined withendothelial cells which express incorporated genetic material ofinterest, comprising the steps of:a) contacting cultured endothelialcells with media containing an infectious recombinant retrovirus havinga recombinant genome comprised of the genetic material of interest; b)maintaining the cultured endothelial cells with media containing aninfectious recombinant retrovirus, under conditions appropriate forinfection of the endothelial cells by recombinant retrovirus; and c)lining the inner surface of a prosthetic vessel with the endothelialcells infected in step (b) under conditions appropriate for maintenanceof the endothelial cells.
 30. Transduced endothelial cells containingstably maintained genetic material of interest, said transducedendothelial cells having been engineered to express the polypeptide orprotein product encoded by the genetic material of interest in vivo,wherein said genetic material comprises a retroviral vector, whereinsaid retroviral vector comprises in operable combination: 5' LTRsequence and 3' LTR sequence derived from a retrovirus of interest, aninsertion site for a gene of interest, and said vector does not containa complete gag, env, or pol gene.
 31. Transduced endothelial cells ofclaim 30 in which said genetic material of interest is selected from thegroup consisting of: DNA which is present in and expressed by normalendothelial cells; DNA which does not normally occur in endothelialcells; DNA which normally occurs in endothelial cells but is notexpressed in them at levels which are biologically significant; and anyDNA which can be modified so that it can be expressed in endothelialcells.
 32. Transduced endothelial cells of claim 31, further comprisingDNA which encodes at least one selectable marker.
 33. Transducedendothelial cells of claim wherein said genetic material of interestencodes a protein or polypeptide selected from the group consisting of ahormone, a receptor, an enzyme and a polypeptide.
 34. Transducedendothelial cells of claim 31, wherein said genetic material of interestencodes human parathyroid hormone, tissue plasminogen activator, FactorVIII, low density lipoprotein receptor or beta-galactosidase. 35.Transduced human endothelial cells having genetic material of interestintegrated therein, the transduced human endothelial cells having beenengineered to express the polypeptide or protein encoded by said geneticmaterial of interest in vivo.
 36. Transduced human endothelial cells ofclaim 35, wherein said genetic material of interest is selected from thegroup consisting of: DNA which is present in and expressed by normalendothelial cells; DNA which does not normally occur in endothelialcells; DNA which normally occurs in endothelial cells but is notexpressed in them at levels which are biologically significant; and anyDNA which can be modified so that it can be expressed in endothelialcells.
 37. Transduced human endothelial cells of claim 36, additionallycomprising DNA which encodes at least one dominant selectable marker.38. Transduced human endothelial cells of claim 36 in which the geneticmaterial of interest encodes a protein or polypeptide selected from thegroup consisting of: a hormone, a receptor, an enzyme, and apolypeptide.
 39. Transduced human endothelial cells of claim 38 whereinsaid genetic material of interest encodes human parathyroid hormone,tissue plasminogen activator, Factor VIII, low density lipoproteinreceptor, or beta-galactosidase.
 40. Cultured endothelial cellscontaining integrated DNA encoding a selected protein not made bynaturally-occurring endothelial cells and DNA encoding a selectablemarker.
 41. Transduced endothelial cells that have been engineered toexpress genetic material of interest integrated therein, wherein saidgenetic material of interest was reverse transcribed from RNA which wasprovided as part of a retroviral vector.
 42. Transduced humanendothelial cells that have been engineered to express genetic materialof interest integrated therein, wherein said genetic material ofinterest was reverse transcribed from RNA which was provided as part ofa retroviral vector.
 43. A method of making transduced endothelial cellswhich express integrated genetic material of interest encoding at leastone protein of interest or at least one polypeptide of interest,comprising the steps of:a) contacting endothelial cells with mediacontaining an infectious recombinant retrovirus having a recombinant RNAgenome comprising sequence corresponding to the DNA of interest whereinsaid retrovirus was derived from a retroviral vector selected from thegroup consisting of α-SGC and MFG; and b) maintaining said endothelialcells and the media containing the infectious recombinant retrovirusunder conditions appropriate for infection of the endothelial cells byrecombinant retrovirus, thereby producing transduced endothelial cells.44. A method of claim 43 wherein said infection of the endothelial cellsoccurs in vitro.
 45. A method of producing a confluent layer ofendothelial cells lining the inner surface of a synthetic prostheticvessel, comprising applying to the inner surface of the vesselendothelial cells comprising incorporated genetic material encoding anendothelial cell mitogen, under conditions appropriate for endothelialcells proliferation wherein said genetic material comprises a retroviralvector selected from the group consisting of α-SGC and MFG.
 46. A methodof claim 45, wherein said genetic material encodes endothelial cellgrowth factor.
 47. A method of producing a prosthetic vessel havingendothelial cells which express incorporated genetic material ofinterest on the inner surface of said vessel, comprising lining theinner surface of said vessel with endothelial cells produced by themethod of claim
 44. 48. Transduced endothelial cells of claim 30 whereinsaid vector further comprises a portion of a gag gene coding sequence.49. Transduced endothelial cells of claim 48 wherein said gag codingsequence comprises a splice donor site and a splice acceptor site,wherein said splice acceptor site is located upstream from an insertionsite for said gene of interest.
 50. Transduced endothelial cells ofclaim 49 wherein said vector further comprises a gag transcriptionalpromoter functionally positioned such that a transcript of a nucleotidesequence inserted into said insertion site is produced, wherein saidtranscript comprises gag 5' untranslated region.
 51. Transducedendothelial cells of claim 50 wherein said endothelial cells are humanendothelial cells.
 52. Transduced endothelial cells of claim 51 in whichthe genetic material of interest is selected from the group consistingof: DNA which is present in and expressed by normal endothelial cells;DNA which does not normally occur in endothelial cells; DNA whichnormally occurs in endothelial cells but is not expressed in them atlevels which are biologically significant; and any DNA which can bemodified so that it can be expressed in endothelial cells. 53.Transduced endothelial cells of claim 50, wherein the genetic materialof interest encodes a hormone, a receptor, an enzyme or a polypeptide.54. Transduced endothelial cells of claim 52 wherein the geneticmaterial of interest encodes human parathyroid hormone, tissueplasminogen activator, Factor VIII, low density lipoprotein receptor, orbeta-galactosidase.